U.S. patent number RE38,047 [Application Number 09/800,499] was granted by the patent office on 2003-03-25 for hemicellulase supplement to improve the energy efficiency of hemicellulose-containing animal feed.
This patent grant is currently assigned to ChemGen Corporation. Invention is credited to David M. Anderson, Douglas W. Fodge.
United States Patent |
RE38,047 |
Fodge , et al. |
March 25, 2003 |
Hemicellulase supplement to improve the energy efficiency of
hemicellulose-containing animal feed
Abstract
Soil microorganisms are obtained that produce a hemicellulase
which is particularly useful in increasing the available energy
content of hemicellulosic foodstuffs. These microorganisms can be
cultured per se or can be used as sources of genetic information
with which to engineer other microorganisms to produce the enzyme.
Thus, commercially useful quantities of native or recombinant
hemicellulase can be produced with cultures consisting essentially
of microorganisms that produce the enzyme. The hemicellulase can
then be employed in a feed composition containing complex
carbohydrates which the enzyme degrades, enhancing the nutritional
value of the composition.
Inventors: |
Fodge; Douglas W. (Rockville,
MD), Anderson; David M. (Rockville, MD) |
Assignee: |
ChemGen Corporation
(Gaithersburg, MD)
|
Family
ID: |
24109453 |
Appl.
No.: |
09/800,499 |
Filed: |
March 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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529327 |
May 29, 1990 |
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Reissue of: |
912675 |
Jul 14, 1992 |
05429828 |
Jul 4, 1995 |
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Current U.S.
Class: |
426/18; 426/2;
426/28; 426/44; 426/53; 426/61; 426/630; 426/634; 426/64 |
Current CPC
Class: |
C12Y
302/01078 (20130101); C12Y 302/01101 (20130101); C12N
9/2437 (20130101); C12N 9/2494 (20130101); A23K
10/10 (20160501); A23K 20/189 (20160501); A23L
29/06 (20160801); A23L 29/262 (20160801) |
Current International
Class: |
A23K
1/00 (20060101); A23K 1/165 (20060101); A23L
1/03 (20060101); A23L 1/052 (20060101); A23L
1/0534 (20060101); C12N 9/24 (20060101); A23L
001/10 () |
Field of
Search: |
;426/2,18,20,21,28,44,46,53,54,61,64,72,74,618,622,623,625,627,629,630,634,635 |
References Cited
[Referenced By]
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1212826 |
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2602260 |
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DE |
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0285098 |
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Oct 1988 |
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EP |
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2302336 |
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Feb 1975 |
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FR |
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48-77085 |
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Jan 1972 |
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JP |
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53-62848 |
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Jun 1978 |
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JP |
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58-141755 |
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Aug 1983 |
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JP |
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63-269993 |
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Nov 1988 |
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JP |
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63-279761 |
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Nov 1988 |
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JP |
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85/04201 |
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Sep 1985 |
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WO |
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Other References
Novo Enzymes Proces Divison; Energex,; p. 1; May (1989). .
JP 63-209586, beta-mannanose-composition and production thereof,
Dec. 23, 1988, abstract only, Patent Abstracts of Japan.* .
Paul Matsudaira "Sequence from picomole quantities of proteins
electroblotted onto polyvinylidene diflourida membranes," J. Biol.
Chem. 262:10035-38 (1987), p. 30, line 27.* .
Feedstuffs 62:10 (1990), p. 2, line 9.* .
Methods in Enzymology, vol. 160, pp. 180-187 and pp. 368-377.*
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Methods in Enzymology, vol. 160, pp. 10-19.* .
Ebisu et al., "Conserved structures of cell wall protein genes
etc.", J. Bacteriol. (173(3):1312-1320 (1990), p. 19.* .
A. Araujo et al., "Hemicellulases of Bacillus species: preliminary
comparative studies etc." J. Applied Bacteriol 68:253-261 (1990),
p. 13, line 13.* .
Almin et al., "Extracellular enzyme system utilized by the Fungus
Sporotrichum pulverulentun etc." Eur. J. Biochem. 51:207-211
(1975).* .
Almin and Eriksson, "Enzymic degradation of polymers, II
Viscometric determination of cellulase activity in absolute terms,"
Biochim. Biophys. Acta 139:248-253 (1967).* .
Almin and Eriksson, "Enzymic degradation of polymers, I Viscometric
determination of cellulase activity in absolute terms," Biochim,
Biophys. Acta 139:238-247 (1967).* .
Methods in Enzymology, vol. 160, pp. 522-633.* .
Hamamoto et al., "Nucleotide sequence of the xylanase A gene of
alkalophilic Bacillus sp. strain C-125," Agric. Biol. Chem.
51(3):953-55 (1987).* .
Esteban et al., "B-D-Xylanases of Bacillus Circulans WL-12," Can.
J. Microbiol. 28:733-39 (1982).* .
Hansen et al., "Alginase enzyme production by Bacillus circulans,"
Appln. Environ. Microbiol. 47(4):704-709 (1984).* .
Patel et al., "The effect of autoclaving and enzyme supplementation
of guar meal on the performance etc." Poultry Sci. 64:1148-1156
(1985).* .
Al-Ani et al., "Effect of chemical pretreatments on the
fermentation and ultimate digestibility etc.", J. Sci. Food Agric.
42:19-28 (1988).* .
S.V.S. Verma et al., "Guar meal in diets for broiler chickens,"
British Poultry Sci. 23:95-105 (1982).* .
Ray et al., "The effect of a purified guar degrading enzyme on
chick growth", Poultry Sci. 61:488-494.* .
Trank et al., "Isolated soy protein production using temperature
sensitive gels", Food Technology, Jun. 1989, pp. 78-83.* .
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enzymatically deamidated soy proteins", J. Food Sci. 54(3):598-601
(1989).* .
Teves et al., "Nutritional Value of Copra Meal Treated with
Bacterial Mannanase in Broiler Diets", In: Recent Advances in
Biotechnology and Applied Biology, Proceedings of Eighth
International Conference on Global Impacts of Applied Microbiology
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Aug. 1-5, 1988, Hong Kong, Chinese University Press, pp. 497-507
(1988)..
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Primary Examiner: Wong; Leslie
Attorney, Agent or Firm: Foley & Lardner
Parent Case Text
This application is a continuation of application Ser. No.
07/529,327, filed May 29, 1990, now abandoned.
Claims
What is claimed is:
1. A feed composition .[.comprising.]. .Iadd.consisting essentially
of .Iaddend.(A) protein, vitamins and minerals; (B) a .[.source of
carbohydrates, comprising a mannan-containing hemicellulose.].
.Iadd.carbohydrate source .Iaddend.selected from the group
consisting of soybeans, corn, and alfalfa.Iadd., wherein said
source comprises a mannan-containing hemicellulose.Iaddend.; and
(C) a mannanase that catalyzes the degradation of said
mannan-containing hemicellulose, where the addition of said
mannanase decreases the ratio of feed to gain, or increases weight
gain, during growth of an animal fed said feed composition,
relative to said animal fed on an identical feed composition absent
said mannanase.
2. A feed composition according to claim 1, wherein said mannanase
is a Bacillus mannanase.
3. A feed composition according to claim 1, wherein said
.[.group.]. .Iadd.carbohydrate source .Iaddend.consists of soybeans
and alfalfa.
4. A feed composition according to claim 1, wherein said
composition is for a monogastric animal.
5. A feed composition .[.comprising.]. .Iadd.consisting essentially
of .Iaddend.soybean meal and a mannanase component that decreases
the ratio of feed to gain, or increases weight gain, during growth
of an animal fed said feed composition, relative to said animal fed
an identical feed composition absent said mannanase.
6. A feed composition according to claim 5, wherein said mannanase
is a Bacillus mannanase.
7. A nutritive method for a monogastric animal, comprising the
steps of (A) preparing a feed composition .[.comprised.].
.Iadd.consisting essentially .Iaddend.of .Iadd.(i)
.Iaddend.protein, vitamins.Iadd., .Iaddend.and minerals, and
.[.further comprising.]. .Iadd.(ii) .Iaddend.a carbohydrate source
.[.comprised of mannan-containing hemicellulose.]. selected from
the group consisting of soybeans, corn, and alfalfa.Iadd., wherein
said source comprises a mannan-containing hemicellulose.Iaddend.;
and (B) incorporating into said composition a mannanase that
catalyzes the degradation of said mannan-containing hemicellulose,
where the addition of said mannanase decreases the ratio of feed to
gain, or increases weight gain, during growth of an animal fed said
feed composition, relative to said animal fed on an identical feed
composition absent said mannanase.
8. A method according to claim 7, wherein said .[.group.].
.Iadd.carbohydrate source .Iaddend.consists of soybeans and
alfalfa.
9. A method according to claim 7, wherein step (B) comprises (i)
producing a mixture containing said mannanase and said composition
and then (ii) pelletizing said mixture under conditions including
temperatures of at least 60.degree. C.
10. A method according to claim 9, wherein said carbohydrate source
is soybeans.
Description
BACKGROUND OF THE INVENTION
Different enzymes are categorized as a specific type of
hemicellulase--a glucanase, a xylanase or a mannanase, for
example--based on an ability to catalyze the hydrolysis of
heteropolysaccharides composed of glucan, xylan or mannan,
respectively. It is known that enzymes that effect hydrolysis of
mannans, such as a galactan or a glucomannan, are produced by
various microorganisms, including bacteria and fungi, and that they
also occur in some animals and in numerous plants. Among the
microorganisms that produce such mannanases are species of
Aeromonas, Aspergillus, Streptomyces, Rhodococcus and Bacillus. See
160 METHODS IN ENZYMOLOGY Part A, Sect. II (1988).
Hemicellulases have been employed commercially in the processing of
coffee, chocolate, cocoa, tea and cereals. The primary advantage
gained by using a hemicellulase in this regard is a reduction in
solution viscosity which allows for more inexpensive processing of
food products. Thus, hemicellulases are used to clarify fruit
juices, to reduce the viscosity of slurries or purees, to liquefy
certain cell wall solids, and to modify taste. But if the available
energy content of human food and animal feed products could be
increased, particularly in animal feed, there would be
opportunities for cost savings. The successful use of
glucanase-treated barley as a corn substitute in avian diets is one
such example. See Feedstuffs 62: 10 (1990).
Hemicellulosic materials such as alfalfa, coconut residue, guar,
locust bean gum, carob bean gum, cassava, copra and soybeans are
common constituents of food and feed products. Soybean derivatives
comprise a substantial proportion of the ingredients of tofu for
human consumption, for example, and soybean protein is used in many
feeds for dogs and cats, swine, fish and chickens. Soybean meal may
comprise as much as 25% of the feed for baby chickens. The feed
rations for chickens, such as broilers, are complex mixtures,
formulated from a number of components on a least cost basis. The
rations are required in very large volumes. As a result, costly
storage facilities for the feed components are necessary for the
blending operations. The feed components are blended to provide an
optimal nutritional mixture of protein, essential amino acids,
minerals, vitamins and calories (that is, an energy source).
Soybean meal has been found to be a preferred concentrated source
of protein with amino acids and, while not considered a primary
energy source such as yellow corn, it supplies about 20% of the
energy requirements of broiler chickens.
Although soybean meal provides some carbohydrates and oils which
yield energy, approximately 10% of its total carbohydrate content
is comprised of galactans and pentosans. These carbohydrates are
not absorbed to any appreciable degree by monogastric animals as
the animals are unable to digest them rapidly enough to obtain the
appropriate monosaccharide for further biochemical oxidation. One
approach to increasing the energy content of soybean meal would be
to reduce the galactans and pentosans to low molecular weight
oligosaccharides or monosaccharides like glucose or similar
carbohydrate components which can be more easily metabolized by
monogastric animals.
A need therefore exists for a way to increase the available energy
content of the hemicellulosic component of food and feedstuffs by
converting mannan-containing hemicellulose components therein into
lower molecular weight carbohydrates which can be metabolized by
monogastric animals.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a
hemicellulase-containing composition that, because it also contains
a novel hemicellulase, has an enhanced energy content when
consumed.
It is also an object of the present invention to provide a method
of producing nutritive materials that contain complex carbohydrates
but that nevertheless are usable by a human or monogastric
animal.
In accomplishing these and other objects, there has been provided,
in accordance with one aspect of the present invention, a
consumable composition comprising (A) protein, vitamins and
minerals nutritionally suitable for a monogastric animal or a
human; (B) a source of carbohydrates, comprising a
mannan-containing hemicellulose; and (C) an enzyme that catalyzes
the degradation of said mannan-containing hemicellulose, wherein
the enzyme has a pH profile for its activity in catalyzing said
degradation that ranges from about pH 4.5 to about pH 11. In a
preferred embodiment, the consumable composition is for human
consumption, i.e., its constituents meet applicable regulatory
requirements for human food, as opposed to animal feeds. In another
preferred embodiment, by contrast, the composition is for a
monogastric animal and, hence, need meet only those requirements
which apply to animal feeds.
In accordance with another aspect of the present invention, a
composition containing the above-described components described has
been provided that includes, as component (C), an enzyme that is
active in catalyzing the degradation of mannan-containing
hemicellulose under conditions where both (1) pH is in the range of
8-11 and (2) temperature is at least 60.degree. C.
In accordance with still another aspect of the present invention, a
consumable composition is provided that comprises soybean meal and
an enzyme component that consists essentially of a Bacillus
hemicellulase having a pH profile, relative to its activity in
catalyzing degradation of hemicellulose, that ranges from about pH
4.5 to about pH 11. In one preferred embodiment, the aforementioned
pH profile peaks between about pH 7 and about pH 9.
In accordance with yet another aspect of the present invention, a
nutritive method has been provided that comprises the steps of (A)
preparing a consumable composition comprised of protein, vitamins
and minerals nutritionally suitable for a monogastric animal or
human, and further comprising a carbohydrate source comprised of
mannan-containing hemicellulose; and (B) incorporating into that
composition an enzyme that catalyzes the degradation of the
mannan-containing hemicellulose, wherein the enzyme has a pH
profile for its activity in catalyzing said degradation that ranges
from about pH 4.5 to about pH 11, such that said enzyme renders the
mannan-containing portion of said hemicellulose usable by a
monogastric animal or a human. In a preferred embodiment, the
aforementioned source of carbohydrates is vegetable matter selected
from the group consisting of soybeans, alfalfa, guar, locust bean
gum, carob bean gum, cassava, copra and coconut residue, and more
preferably soybeans and alfalfa. In another preferred embodiment,
step (B) comprises producing a mixture containing the enzyme and
the composition and then pelletizing that mixture.
Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It
should be understood, however, that the detailed description and
the specific examples, while indicating preferred embodiments of
the invention, are given by way of illustration only since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
In drawings which illustrate embodiments of the invention,
FIG. 1 is a graph which plots the activity of a hemicellulase
within the present invention versus time (pH 9.0) at 60.degree.,
75.degree. and 90.degree. C., respectively.
FIG. 2 is a graph that compares the pH profile of enzymatic
activity for a hemicellulase of the present invention versus a
known, commercially available enzyme.
FIG. 3 presents representative data showing the reduction in
viscosity in relation to the amount of time the diluted enzyme was
incubated with the guar solution.
FIG. 4 presents data showing the average weight of chickens
maintained for the first 21 days of life on a control diet versus
the average weight of chickens maintained on a diet of
hemicellulase-containing animal feed. The ratio of the amount of
feed consumed to the amount of weight gained (feed/gain) for both
populations is also presented.
FIG. 5 presents data showing the average weight of chickens
maintained for 46 days of life on a control diet versus the average
weight of chickens maintained on a diet of hemicellulase-containing
animal feed. The ratio of the amount of feed consumed to the amount
of weight gained (feed/gain) for both populations is also
presented.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
It has been discovered that a hemicellulase capable of catalyzing
the degradation of hemicellulose in a food material, with a
resulting increase in available energy content of that material,
can be obtained from microorganisms which produce the enzyme.
Microorganisms having the ability to produce a hemicellulase within
the present invention can be isolated by conventional methods from
the soil, where they comprise a reproducible, finite subsection of
indigenous microflora. Hemicellulase-producing microorganisms can
also be produced by transforming another microorganism, such as B.
subtilis or B. brevis, with hemicellulase-encoding DNA obtained,
via standard recombinant DNA techniques, from such indigenous soil
microflora.
In cultures consisting essentially of microorganisms that produce a
hemicellulase of the present invention, commercially significant
quantities of the enzyme can be obtained by isolation techniques
well known in the art. In this description, a "culture consisting
essentially of a specified type of microorganism," such as a
hemicellulase-producing microorganism, is a culture that is
comprised primarily of microbes of that type, to the extent that
the salient functional property or properties of the culture are
determined by those microbes. But other types of microorganisms may
be present, for example, in a culture consisting essentially of a
hemicellulase-producing microorganism, so long as the other types
do not interfere significantly with hemicellulase production by the
culture.
Microorganisms which produce a hemicellulase within the present
invention can be isolated from soil samples collected from diverse
geographical areas. The soil samples are collected primarily from
the top two inches of soil and cultured in selective enrichment
media.
Techniques for selectively isolating microorganisms with certain
desired characteristics are well known in the art. Microorganisms
can be selected for the capacity to produce a particular enzyme by
exposing samples potentially containing such microorganisms to
conditions wherein replication and growth are dependent upon the
capacity to produce the desired enzyme. One widely used method of
selection is exposing a sample containing a large and diverse
population of microorganisms to a medium consisting of a single
carbon source. See 160 METHODS IN ENZYMOLOGY 180-86 (1988). Only
those microorganisms capable of producing enzymes that can degrade
the carbon source will be recovered by this method. Selective
culturing techniques of this sort effectively separate
microorganisms producing the desired enzyme from the myriad of
other microorganisms comprising the normal flora of the habitat
sampled.
In a preferred embodiment of the present invention, aliquots of
soil samples are inoculated into an alkaline culture medium,
wherein hemicellulose serves as the sole carbon source. Dilutions
of the sub-cultures grown in the same medium are plated out onto a
solid medium which also contains hemicellulose as the sole carbon
source. Morphologically distinct colonies are isolated and then
screened for hemicellulase activity. In a particularly preferred
embodiment of this invention, a selective enrichment broth at a pH
range of 99.5, wherein guar is the sole carbon source is inoculated
with a sample of soil, incubated at 37.degree. C. and aerobically
agitated. After incubation, further dilutions of the initial
culture are made using the selective enrichment broth. After
several passages, a dilution series of the most dilute broth
culture is made in normal saline and plated out on a solid medium
which contains guar as the sole carbon source. After incubation at
34.degree. C. for 5-7 days, morphologically distinct colonies are
isolated from the solid medium and screened for hemicellulase
activity.
The initial selection for microorganisms which utilize
hemicellulose as a sole carbon source at an elevated pH results in
the recovery of a finite fraction of all soil microorganisms.
Typically, about 40% of the isolates are characterized by an
ability to produce a hemicellulase of the present invention. A
particularly preferred subgroup of this fraction includes species
of the Gram-positive genus Bacillus.
Exemplary of this preferred subgroup is a strain of B. circulans
designated CMG1240, the general properties of which are summarized
in Table I below. Strain CMG1240 has been deposited, in accordance
with the Budapest Treaty, at the American Type Culture Collection
(Rockville, Md.) under accession No. 55045. The key characteristic
of strain CMG1240, as for any microbial strain of the preferred
subgroup, is its ability to produce in culture medium a
hemicellulase suitable for use according to the present
invention.
Conventional methods for measuring cellulase/hemicellulase
activities are described, for example, in 160 METHODS IN ENZYMOLOGY
180-86 & 368-76 (1988). These methods generally entail exposing
a predetermined amount of a substrate to a predetermined amount of
a enzyme preparation, crude or purified. The rate of conversion of
the substrate to the desired end product is measured under
specified conditions of pH and temperature. Crude enzyme
preparations can be produced by culturing microorganisms in a
suitable medium and then concentrating the enzyme that accumulates
in the broth surrounding the cells using conventional means such as
precipitation or ultrafiltration. The resulting enzyme preparations
can be assayed for specific activity by methods known to the art,
including those that are keyed, respectively, to weight loss of
insoluble substrates, changes in turbidity of polysaccharide
suspensions, an increase in reducing end groups, a decrease in the
viscosity of polysaccharides such as .beta.-mannans, colorimetric
determinations, measurements of clearance zones in
polysaccharide-agar, or polarography.
TABLE I
a. Morphology (1) bacillus shape; (0.8.times.1.7-2.3).mu.m (2)
occurs predominately in singles and pairs (3) capable of forming
endospores (4) Gram-positive (also appears Gram-variable or
Gram-negative)
b. Growth Conditions (1) Tryptic soy agar plate culture: colonies
are irregular, convex, smooth, undulate, butyrous, opaque, and 2 mm
in diameter (72 hours, 37.degree. C.)
c. Biochemical Properties: (1) aerobic (2) catalase positive (3)
oxidase negative (4) non-motile (5) growth temperature:
30.degree.-50.degree. C., maximum growth rate occurring at
41.degree. C. (6) growth pH (cultured at 34.degree. C. at 350 rpm
for 10 hours in a basal glucose-mineral salts medium): 7.0-8.5,
maximum growth rate occurring at pH 7.5 (7) casein and gelatin
decomposition: negative (8) starch hydrolysis: positive (9) growth
in 1-5% NaCl: positive (10) carbon sources utilized include:
D-glucose, D-galactose, D-fructose, D-xylose, lactose, maltose,
sucrose, .alpha.-cyclodextrin, dextrin, glycogen,
N-acetylglucosamine, L-arabinose, cellobiose, gentiobiose,
lactulose, mannitol, D-mannose, D-melibiose, D-trehalose, turanose,
.alpha.-ketobutyric acid, uridine, and m-inositol (11) hydrogen
sulfide production: negative (12) indole production: negative (13)
utilization of citrate: negative (14) urease: negative (15)
Voges-Proskauer: negative (16) phenylalanine deaminase: negative
(17) lysine decarboxylase: negative (18) ornithine decarboxylase:
negative (19) arginine dehydrolase: negative
In accordance with the present invention, soil isolates are
screened for hemicellulase production by culturing isolated
colonies of the microorganisms in a hemicellulose-containing broth
medium. After incubation, the broth medium is centrifuged and the
resulting supernatant is filtered to remove cells and to produce a
crude preparation of hemicellulase. The enzyme is added to
alkaline, viscous preparations of a preferred hemicellulose and the
degree of liquefaction of the substrate over time is measured.
It is preferred that soil isolates are cultured in a broth medium
wherein a mannan-containing hemicellulose is the sole carbon
source. After incubation, the broth medium is centrifuged and the
resulting supernatant is filtered. The resulting crude enzyme
preparation is added to a highly viscous preparation of a
mannan-containing hemicellulose at a pH in the range of 8-11. The
relative activity of the enzyme is determined by the amount of time
required to liquefy the hemicellulose.
It is particularly preferred that soil isolates are cultured, as
described above, in a selective enrichment medium wherein guar is
the sole carbon source. After incubation, the culture is
centrifuged and the resulting supernatant filtered to recover crude
enzyme. The crude enzyme is then introduced into tubes containing a
cross-linked guar preparation, with metal ions added to enhance the
viscosity of the preparation, at a pH in the range of 9-9.5. After
incubation for at least one hour at 39.degree.-40.degree. C.,
enzyme activity is measured by determining the viscosity of a
enzyme/guar solution using conventional viscosimetric procedures.
See, e.g., Biochim. Biophys. Acta 139: 238 & 248 (1967); Eur.
J. Biochem. 51: 207 (1975).
A hemicellulase obtained as described above will generally display
an activity characterized by a pH profile--the curve defined by the
relationship between a given enzymatic activity and pH--that ranges
over some 6.5 pH units, between minima at about pH 4.5 and about pH
11, respectively. Such a pH profile is highly distinctive when
compared, for example, to corresponding profiles for known Bacillus
mannanases, which are virtually inactive at above about pH 8. See
Araujo & Ward, J. App. Bacteriol., 68: 253-61 (1990).
A hemicellulase used in the present invention preferably has a pH
profile with a peak (i.e., the portion of the profile where
activity is greatest) that is in a range from about pH 9 to about
pH 7. It is also preferred that the enzyme display significant
biological activity under conditions characterized both by high
alkalinity and by elevated temperatures. Such a suitable enzyme
would display significant activity, for example, when pH is in the
range of 8-11 and the temperature is 60.degree. C. or higher.
A hemicellulase particularly preferred for use according to the
present invention is an endo-.beta.-D-mannanase having the
following properties:
(1) Activity: A preferred enzyme acts on hemicellulosic materials
containing mannan carbohydrates like galactomannan, glucomannan and
mannan. Activity in this context can be measured in the following
way. An aqueous suspension containing 1.0% guar is used as a
substrate, and 2 mL of 2M glycine is added to 16.0 mL of the
substrate. The mixture is mixed thoroughly and then preheated to
38.degree. C. Enzyme is added to the substrate, mixed well and then
the viscosity of the enzyme/guar solution is measured using a
simple approach, as by timing the charging and discharging of a
pipette with stopwatch, or more complex devices like Brookfield or
Fann viscometers. A standard curve is prepared using a commercially
available hemicellulase enzyme with known activity such as Miles
B1500. Grams or units of the commercially available hemicellulase
are plotted against the time required to change viscosity and the
new hemicellulase is compared to that of the commercial
product.
(2) Substrate specificity: The enzyme degrades relatively simple
carbohydrate polymers like guar gum, which is a polymer of
D-mannose linked .beta.-1,4 with D-galactose, and locust bean gum,
as well as more complex, mannan-containing carbohydrates, for
example, from soybean and alfalfa. Other suitable substrates will
include mannan-containing coconut residue, carob bean gum, cassava,
copra, and chemically-modified forms of guar.
(3) Optimum pH: The optimum pH of the enzyme is about 7.0 or
slightly above, e.g., in the range 7.1 to 7.5. The enzyme is stable
in a pH range of about 4.5 to 11 (see FIG. 2).
(4) Optimum temperature: The optimum temperature for enzyme
activity is 40.degree. C., but enzyme activity is observed at
temperatures ranging from 20.degree. to 90.degree. C. As shown in
FIG. 1, the enzyme exhibits significant activity at temperatures
ranging from 60.degree. C. to 90.degree. C.
(5) Deactivation by Temperature and pH: At pH 9.0 the enzyme
retains 50% of its maximum activity after 5.5 hours at 60.degree.
C., after 45 minutes at 75.degree. C., and after 15 minutes at
90.degree. C. (see FIG. 1).
(6) Molecular Weight: After purification to homogeneity, the enzyme
has a molecular weight of about 32,000 as determined by
SDS-polyacrylamide gel electrophoresis. To obtain a crude enzyme
preparation from culture broth, cells and debris are first removed
with a 0.1-micron filter. The enzyme is then concentrated with a
10,000 molecular weight cut-off membrane. However, once the enzyme
becomes quite concentrated, a good proportion of the enzyme is able
to pass through the membrane. The enzyme in this final permeate can
be concentrated with a 5,000 molecular weight cut-off membrane or
precipitated with three volumes of acetone. After the precipitate
is centrifuged, the supernatant is siphoned off and the pellet
resuspended in 50 mM phosphate buffer. This concentrate is dialyzed
versus 10 mM phosphate buffer (pH 7.0) and then applied to a
DEAE-Sephacel column. The enzyme is eluted with a solution gradient
of sodium chloride of increasing ionic strength ranging from
0-1.25M in 50 mM phosphate buffer (pH 7.0). Fractions are collected
and tested for enzyme activity. The fractions exhibiting maximum
activity are pooled and examined using SDS-polyacrylamide gel
electrophoresis and compared to protein molecular weight
standards.
Pursuant to the present invention, commercially useful quantities
of hemicellulase can be produced by culturing the foregoing
hemicellulase-producing microorganisms using conventional
fermentation techniques. In this context, "fermentation" is used
broadly to refer to any controlled microbial action by which useful
products are obtained from the substrate(s) of that action. In
accordance with the present invention, fermentation can be carried
out in a stirred-tank reactor. This type of reactor is essentially
a closed cylindrical tank containing agitators, baffles, heat
exchange coils, and automatic controls for temperature, air flow,
pressure, pH and foaming. A fermenter of this sort would be charged
with essential nutrients and a hemicellulase inducer. After
sterilization, the reactor is inoculated with a culture consisting
essentially of microorganisms that have been selected for their
capacity to produce hemicellulase as described above.
Continuous culture can increase fermenter productivity by
eliminating the downtime of batch cultures. But it is sometimes
difficult to maintain sterility in large scale continuous cultures.
Accordingly, batch fermentations which utilize the bacteria of the
present invention are preferred.
In a preferred embodiment of this invention, a Gram-positive
Bacillus strain, such as B. circulans CMG1240, is utilized to
produce commercially useful quantities of the enzyme. A fermenter
charged with a medium consisting of industrial-grade nutrients,
glycerol (carbon source) and a mannan-containing hemicellulose,
such as guar gum, is inoculated with a culture consisting
essentially of a Gram-positive Bacillus strain as described above.
The fermenter is maintained at about 35.degree. C. (500-1,000 rpm
agitation) for about 12 hours. The hemicellulose, such as guar, is
added to the fermenter during the growth phase to further induce
enzyme production. The fermentation is harvested after about 1 to
7.5 hours, and then enzyme activity is measured, for example, by
viscometric means.
Enzyme activity of the fermentation product is assayed by
centrifuging aliquots of the fermentation broth and testing the
resultant supernatant. Aliquots of the supernatant (or dilutions
thereof) are added to a guar solution and any reduction in the
viscosity of the enzyme/guar solution is measured routinely by
recording the time required for a constant bore pipette to
discharge a predetermined volume [see item (1) under the preferred
hemicellulase properties discussed above].
The enzyme is recovered from the fermentation broth by filtration
through a 0.5-micron filter to remove cells followed by
ultrafiltration using a 5,000 or 10,000 molecular weight cut-off
ultrafiltration membrane. The concentrate is combined with three
volumes of acetone at about 4.degree. C. to precipitate the enzyme.
The precipitate is allowed to settle for approximately 24 hours
before the supernatant is siphoned off. The precipitate is then
centrifuged at about 4.degree. C. and the resultant pellet
resuspended in phosphate buffer to form a paste.
In an alternative method for producing a hemicellulase within the
present invention, DNA encoding hemicellulase could be isolated and
used, via known procedures, to transform a suitable host organism
such that the enzyme is produced by the recombinant host in
commercially useful amounts. Hemicellulase-encoding DNA could be
isolated by screening nucleic acid libraries generated from
microorganisms expressing a hemicellulase according to the present
invention. See, e.g., Ausubel et al., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY at .sctn..sctn.5 and 6, John Wiley and Sons, New
York (1987, 1990) ("Ausubel"). Such libraries would be screened by
means of oligonucleotide probes that are complementary to a
polynucleotide encoding, for example, a portion of the N-terminus
of a hemicellulase within the present invention. Illustrative of
such an N-terminal portion is the amino acid sequence
Ala-Ser-Gly-Phe-Tyr-Val-Xxx-Gly-Thr-Ile-Leu-Xxx-Asp-Ser-Thr-Gly-Asn-Pro-Ph
e-Lys-Ile-Xxx-Gly-Xxx-Asn [Xxx denotes an indeterminant amino
acid], as discussed in Example 12 infra. See Ausubel at
.sctn.6.
Alternatively, other portions including or adjacent to the
endogenous coding sequence of a hemicellulase according to the
present invention can be used, when isolated using a probe as a
template for generating other probes useful for isolating a
hemicellulase-encoding polynucleotide according to the present
invention, e.g., based on the N-terminal sequence described above.
Such a probe could be used in a known manner to screen a genomic or
cDNA library as described above, see Ausubel, loc. cit., or to
synthesize polymerase chain reaction (PCR) probes for use in
amplifying a cDNA generated from an isolated RNA which codes for a
hemicellulase of the present invention. Such a cDNA could then be
cloned into a suitable expression vector and employed to transform
a host organism. See Ausubel at .sctn.15.4.
A suitable polynucleotide in this regard would preferably comprise
a nucleotide sequence, corresponding to the desired amino-acid
sequence, that is optimized for the host of choice, as described
below, with regard to codon usage, the initiation of translation,
and the expression of recoverable amounts of a commercially useful
hemicellulase within the present invention. Also, a vector selected
for transforming the chosen host organism with such a
polynucleotide molecule should allow for efficient maintenance and
transcription of the sequence encoding the polypeptide. Such a
vector is readily available or derivable from commercial sources,
and is suited to a particular host cell employed for expressing a
hemicellulase according to the present invention. For vectors
exemplary of those suitably used to the present invention, see
Ausubel at .sctn..sctn.2-4.
Host cells suitable for expressing a hemicellulase according to the
present invention would include prokaryotic or eukaryotic cells,
for example, bacterial, algal, yeast, insect, mammalian and human
cells. Thus, among host cells suitable for the present invention
would be microorganisms of the genera Aeromonas, Aspergillus,
Bacillus, Escherichia, Kluyveromyces, Pichia, Rhodococcus,
Saccharomyces and Streptomyces. More specifically illustrative of
the suitable host microorganisms would be the bacteria E. coli, B.
subtilis, B. brevis (J. Bacteriol. 172: 1312-20) and B. circulans,
as well as the yeast S. cerevisiae strain X2181-1B, with genotype a
trp1 gal1 ade1 his2 (available from the Yeast Genetic Stock Center,
Berkeley, Calif.); strain ATCC 52683, with genotype ade1 trp1 metl4
ura3 (available from the American Type Culture Collection,
Rockville, Md.); and strain ATCC 46183, with genotype a his1 trp1
(also available from the American Type Culture Collection). Host
cells comprising a polynucleotide encoding a hemicellulase
according to the present invention can be grown under conditions
that provide expression of a hemicellulase of the present invention
in recoverable or commercially useful amounts. See, e.g., Ausubel
at .sctn..sctn.1 & 13.
Many sources of vegetable matter such as soybeans are rich in
energy containing proteins and carbohydrates and are frequently
utilized as a component of animal feed. Soybeans are also widely
used as components of food products for human consumption.
Galactans are a major component of the total carbohydrate content
of commercial soybean products, however, and do not contribute
significantly to the available energy content of soybean meal when
employed as a component of feed for monogastric animals because
these carbohydrates are not easily digested, and consequently, they
are not absorbed. In order to exploit their energy content,
galactans must be reduced to smaller molecular weight carbohydrates
which can be absorbed and metabolized by monogastric animals and
humans.
In accordance with the present invention, a hemicellulase
possessing the above-mentioned properties can be employed to
degrade galactans and other mannan carbohydrates present in
hemicellulosic constituents of food and animal feedstuffs,
especially those containing complex carbohydrates from soybeans. It
is preferable that the enzyme component included in a consumable
composition of present invention should consist essentially of a
hemicellulase, particularly a Bacillus hemicellulase, as described
above. In this regard, "consisting essentially" means that the
enzyme component displays a hemicellulose-degrading activity which
is determined principally by the aforementioned hemicellulase.
Other enzymes, possibly with other activities, may be present,
however, so long as they do not unduly interfere with the
hemicellulose-degrading activity of the enzyme component.
In a preferred embodiment, an effective amount of hemicellulase
according to the present invention is added to an aliquot of
soybean meal and allowed to dry. The resulting composition is
ground to a fine powder and then mixed with dry animal feed
consisting, for example, of corn and other nutrients.
A mixture of this sort can be processed under conditions,
particularly of heat and pH, that would inactivate conventional
hemicellulases. Thus, the aforementioned soybean-based composition
can be processed through a press mill to form pellets of animal
feed containing hemicellulase, soybean meal and other animal feed
components in predetermined proportions. When maintained on a diet
of the hemicellulase-containing animal feed, chickens on the
average gain more body weight per pound of feed ingested than
chickens maintained on an identical diet absent hemicellulase.
The present invention is further described below by reference to
the following illustrative examples. In those examples, the
following media were employed:
Selective Enrichment Broth (amount/liter) 10.0 g Guar Gum 5.0 g
(NH.sub.4).sub.2 SO.sub.4 pH 9.5 Selective Enrichment Agar
(amount/liter) 2.0 g Guar Gum 1.0 g Na.sub.2 HPO.sub.4 3.0 g
(NH.sub.4).sub.2 SO.sub.4 0.2 g NaCl 0.2 g MgSO.sub.4.7H.sub.2 O
50.0 mg CaCl.sub.2.2H.sub.2 O 1.0 mL Trace Elements Solution I (see
below) 15.0 g Agar Noble 50.0 mM Tris Buffer (pH 9.0) 1.0 mL
Vitamin Solution (see below) Trace Elements Solution I
(amount/liter) 100.0 mg EDTA 230.0 mg ZNSO.sub.4.7H.sub.2 O 180.0
mg MnSO.sub.4.H.sub.2 O 60.0 mg H.sub.3 BO.sub.3 100.0 mg
CuSO.sub.4.5H.sub.2 O 40.0 mg Na.sub.2 MoO.sub.4.2H.sub.2 O 40.0 mg
CoCl.sub.2.6H.sub.2 O 70.0 mg KI 40.0 mg FeSO.sub.4.7H.sub.2 O 0.4
mg NiCl.6H.sub.2 O 8.0 .mu.L 0.1M H.sub.2 SO.sub.4 Vitamin Solution
I (amount/liter) 1.0 g Vitamin B12 1.0 g Riboflavin, B2 1.0 g
Pyridoxine, B6 1.0 g D-biotin 1.0 g Thiamine Hydrochloride 1.0 g
Nicotinic Acid 1.0 g D-Ca-Pantothenic acid Seed Broth
(amount/liter) 7.5 g Glycerol 10.0 g Yeast Extract 2.5 g Corn Steep
Liquor 1.0 g KH.sub.2 PO.sub.4 2.0 g (NH.sub.4).sub.2 SO.sub.4 0.5
g MgSO.sub.4.7H.sub.2 O 1.0 mL Trace Elements Solution II (see
below) pH 7.0-7.5 Fermentation Broth (amount/liter) 20.0 g Glycerol
20.0 g Yeast Extract 5.0 g Corn Steep Liquor 2.0 g KH.sub.2
PO.sub.4 4.0 g (NH.sub.4)SO.sub.4 1.0 g MgSO.sub.4.7H.sub.2 O 2.0
mL Trace Elements Solution II pH 8.5 Trace Elements Solution II
(amount/liter) 20.0 g FeSO.sub.4.7H.sub.2 O 20.0 g
FeCl.sub.3.6H.sub.2 O 0.5 g MnSO.sub.4.H.sub.2 O 50.0 mg
CoSO.sub.4.7H.sub.2 O 10.0 mg CuSO.sub.4.5H.sub.2 O 20.0 g
CaCl.sub.2.2H.sub.2 O 50.0 mg H.sub.3 Bo.sub.3 100.0 mg ZnSO.sub.4
7H.sub.2 O 100.0 mg Na.sub.2 MoO.sub.4.2H.sub.2 O
EXAMPLE 1
Isolation of Hemicellulase-Producing Microorganisms from Soil
Soil samples collected from both a tropical rain-forest and a
temperate garden were added to selective enrichment broth in a 10%
w/v concentration. The cultures were shaken in baffled Erlenmeyer
shake flasks at 37.degree. C. for four days. Four dilutions [1:10,
1:20, 1:800 and 1:50,000] of the initial cultures were made with
fresh selective enrichment broth and incubated at 37.degree. C. for
four days. The 1:50,000 dilution was used to prepare a dilution
series (10.sup.-1 to 10.sup.-8) in 0.85% NaCl which were plated out
on selective enrichment agar and incubated at 34.degree. C. for 5-7
days. Following incubation, isolated colonies were selected from
the plated cultures and streaked for purity three successive times
on a suitable agar medium. Nine isolates from soil collected from a
tropical rain-forest and 24 isolates from soil collected from a
temperate garden were selected as a result of this screening
process.
EXAMPLE 2
Screening of Soil Isolates for Hemicellulase Activity
Each purified isolate from the tropical rain-forest soil was
transferred to a shake flask containing selective enrichment broth
and shaken for 48-72 hours at 34.degree. C. After incubation, the
culture was centrifuged for 20-30 minutes at 10,000 rpm (4.degree.
C.). The resultant supernatant was filtered sequentially through a
0.8- and a 0.45-micron filter to recover crude enzyme free of
microbial cells. Each purified isolate from the temperate garden
soil was transferred to tubes containing selective enrichment broth
and shaken 12-48 hours at 34.degree. C. After incubation, the
cultures were centrifuged and filtered as described above. The
crude enzyme solution was added to tubes containing 5 mL of a
cross-linked guar preparation (5.0 g guar, 2.0 g (NH.sub.4).sub.2
SO.sub.4 and 0.6 g sodium tetraborate per 400 mL water, pH 9.5) and
incubated for at least 1 hour in a water bath at
39.degree.-40.degree. C. To measure enzyme activity, a constant
bore 1 mL pipette was charged with 1.0 mL of the mixture and
allowed to discharge 0.9 mL. A stopwatch was used to measure the
time required for this discharge to occur (hereinafter "drop
time"). A drop time of less than two seconds indicated measurable
amounts of hemicellulase activity. Twelve of the twenty-four
isolates recovered from temperate garden soil, and four of the nine
isolates recovered from tropical rain-forest soil produced
measurable amounts of hemicellulase.
EXAMPLE 3
Production of Commercially Useful Quantities of Hemicellulase
A single, isolated colony of Bacillus circulans (CMG1240) was
inoculated into a 500 mL Erlenmeyer baffled shake (pre-seed) flask
containing 50 mL of brain heart infusion broth. The inoculated
flask was shaken (300 rpm) while incubating for 12-16 hours at
35.degree. C. After incubation, the entire volume of the pre-seed
flask was aseptically transferred to a 4 L Erlenmeyer baffled
shake-flask (seed) containing one liter of seed medium. The
seed-flask was incubated with shaking for 12-16 hours at 35.degree.
C. The entire volume of the seed flask was aseptically transferred
to a 14-liter fermenter containing 9.0 liters of fermentation
broth. The fermenter was maintained at 40.degree. C., <20%
dissolved oxygen and 500-1000 rpm. Guar gum was added to make a
final concentration of 0.05% to the fermenter two or three times
during the growth phase and once (0.5%) during the stationary phase
to induce enzyme production. After 1-7.5 hours, the fermentation
was terminated and enzyme activity assayed according to the
procedure of Example 5. Assay data are shown in FIG. 3.
EXAMPLE 4
Measurement of Hemicellulase Activity to Determine Optimal Culture
Conditions for Hemicellulase Production
Twenty milliliter aliquots of 1% guar in water, mixed for 3 hours
to obtain the maximum viscosity, and 2 mL aliquots of 2M
glycine-NaOH buffer (pH 9.0) were added to a glass-jacketed reactor
maintained at 40.degree. C. by means of a circulating water bath.
Aliquots of the fermentation product were centrifuged in a
Microfuge for 5 minutes and the resultant supernatant was used to
test for enzyme activity. A 100-200 .mu.L sample of the supernatant
was added to the reactor. Enzyme activity was measured as described
in Example 5. Sample enzyme activity was calculated from a standard
curve prepared by plotting the log of the drop time (seconds) of
dilution of a standard hemicellulase (Miles B-1500) versus
incubation time in minutes. The slope (on a linear scale) of the
log plot was calculated and replotted versus grams or "chamber
units" of hemicellulase.
EXAMPLE 5
Assay for Hemicellulase Activity Suitable for Testing Standards and
Purified Enzyme Preparations
In a procedure suitable for screening enzyme activity of standard
hemicellulase or purified enzyme preparations, approximately 16.0
mL of a guar preparation, comprising 1% guar gum in water, and 2.0
mL of 2M glycine (adjusted to pH 9.0 with NaOH), were added to a
100 mL glass jacketed reactor maintained at 38.degree. C. by means
of a circulating water bath. The solution was mixed thoroughly by a
magnetic stirrer after reaching temperature. Viscosity at time zero
was determined by measuring the time required for the solution to
drop from the 1.0 mL mark to the 0.7 mL mark in a constant bore, 1
mL pipette. The initial drop time of the guar solution without
enzyme added was 75 to 90 seconds. After enzyme addition (200-500
.mu.L) to the reactor, the guar/enzyme solution was allowed to
incubate. A time-zero measurement of the solution was immediately
determined. Drop time measurements were performed until either five
measurements had been made or until the drop time was less than 30
seconds. A standard curve was prepared by plotting the log of the
drop time versus the time of incubation using different standard
enzyme dilutions.
EXAMPLE 6
Recovery of Hemicellulase from Fermentation
The procedure described in Example 3 was used in the production of
useful quantities of hemicellulase in each of six 14-liter
fermentations. After the fermentation was terminated, cells were
removed from 60 L of culture broth by ultrafiltration with a
0.1-micron filter. Then, the enzyme was concentrated to a volume of
approximately 3.5 L with a 10,000 molecular weight cut-off
ultrafiltration membrane. The concentrate was combined with three
volumes of acetone at 4.degree. C. to precipitate the
hemicellulase. The precipitate was collected by allowing it to
settle for 24 hours and then siphoning off the supernatant. The
precipitate was then centrifuged at 6000 rpm for 10 minutes at
4.degree. C. The resulting pellet was immediately resuspended in
170 mL of 50 mM potassium phosphate buffer (pH 7.0) to a final
volume of 300 mL. The enzyme activity of the resulting
hemicellulase paste was determined according to the method of
Example 5. The recovered hemicellulase paste had
9.45.times.10.sup.6 chamber units/liter, which was equivalent to
6.3 kg/L of standard commercial hemicellulase (assayed at pH 9.0)
or 1.89 kg total of standard hemicellulase formulation.
EXAMPLE 7
Alternate Process for Recovering Hemicellulase
Hemicellulase was recovered by the method described in Example 6,
wherein methanol was used alternatively to precipitate
hemicellulase. The supernatant was siphoned off and the precipitate
centrifuged as described above. The paste-like precipitate was then
blended with 10 volumes (w/w) of lactose and dried under vacuum at
temperatures up to 40.degree. C.
EXAMPLE 8
Alternate Process for Recovering Hemicellulase
Hemicellulase was concentrated to a volume of approximately 15 L
with a 10,000 molecular weight cut-off ultrafiltration membrane.
Solid ammonium sulfate was slowly added to 75% saturation with
continuous mixing. The precipitate formed in the resulting solution
was harvested by centrifugation at 8,000 rpm for 45 minutes. The
enzyme activity of the resulting pellet was determined according to
the method of Example 5. The recovery yield of enzyme activity was
1.85.times.10.sup.5 units/gram (2.22.times.10.sup.8 total units).
The recovery yield of enzyme activity of the precipitate was 77% of
the total enzyme activity of the 15 L concentrate.
EXAMPLE 9
Purification of Hemicellulase
To produce a more purified product, two precipitation steps were
used to obtain refined enzyme. A concentrate from ultrafiltration
was prepared according to the method described in Example 6. The
concentrate contained 6.39.times.10.sup.6 units/liter, with a
specific activity of 1.11.times.10.sup.3 units/mg. Equal volumes
(2.2 liters) of the concentrate and acetone were mixed to make a
50% (v/v) solution, and the resultant precipitate was then allowed
to settle for 18 hours at 10.degree. C. After the supernatant was
collected, an additional volume of acetone was added to make the
solution 75% (v/v). The resultant precipitate was allowed to settle
for 24 hours at 10.degree. C., after which the supernatant was
first removed by siphoning and finally by centrifugation at 5000
rpm. The precipitate was then dissolved in 222 mL of 50 mM
phosphate buffer (pH 7.0). Activity was determined to be
5.71.times.10.sup.7 units/liter, with a specific activity of
6.17.times.10.sup.3 units/mg. This purification step resulted in a
recovery of 90% of the total activity with only 20% of the protein,
causing a 5.56 fold increase in the specific activity.
EXAMPLE 10
Alternative Hemicellulase Purification Process
An alternative purification process was employed to avoid the
precipitation step of Example 9. The hemicellulase was concentrated
to the desired concentration by ultrafiltration as described in
Example 9. A liquid formulation of the concentrate was then
prepared by adding stabilizing and preservative agents such as
ethylene glycol, or alternatively, glycerol, methanol, ethanol,
sorbitol, sodium benzoate, sodium citrate, methylparaben, sodium
ascorbate or an equivalent.
EXAMPLE 11
Characterization of the Purified Hemicellulase
A broth concentrate was prepared by ultrafiltration; i.e., cells
were removed with a 0.1-micron membrane and the enzyme concentrated
with a 10,000 molecular weight cut-off membrane. When the enzyme
became concentrated, it was observed that some of the hemicellulase
passed through the membrane. This permeate fraction was
reconcentrated with the 10,000 molecular weight cut-off membrane
and then used for further purification. The reconcentrated permeate
was precipitated by the addition of 3 volumes of acetone at
4.degree. C. The supernatant was siphoned off, and the precipitate
resuspended in 50 mM phosphate buffer (pH 7.0) to a final volume of
222 mL. The enzyme concentrate was subsequently dialyzed versus 10
mM phosphate buffer (pH 7.0) to remove any salts. The dialyzed
concentrate was then applied to a DEAE-Sephacel column
(2.8.times.30 cm). Elution of the hemicellulase was carried out by
adding a solution of increasing ionic strength to the column at a
flow rate of 1 mL/minute. The solution consisted of 0-1.25M NaCl in
50 mM phosphate buffer. Ten milliliter fractions were collected and
tested for enzyme activity by measuring viscosity reduction of a 1%
guar solution as described in Example 5. Fractions 96-109, which
showed maximum activity, were pooled and then analyzed (along with
molecular weight marker-proteins) on a 12.5% SDS-polyacrylamide
gel. The results of this analysis indicated that the hemicellulase
was highly purified, and had a molecular weight of 32,000.
Hemicellulase fractions were ultimately purified by electrophoresis
on the same type of SDS-gel and blotted onto Immobilon P PVDF
membrane and stained with coomassie blue by the method of
Matsudaira, J. Biol. Chem. 262: 10035-38 (1987). The hemicellulase
band on the membrane was excised and used for amino acid sequence
analysis. The enzyme was analyzed by Edman degradation using a
Biosystems Gas Phase Sequenator. The N-terminal sequence of the
purified enzyme was characterized as follows:
1 5 10 Ala-Ser-Gly-Phe-Tyr-Val-Xxx-Gly-Thr-Ile-Leu-Xxx- 15 20 25
Asp-Ser-Thr-Gly-Asn-Pro-Phe-Lys-Ile-Xxx-Gly-Xxx-Asn
EXAMPLE 12
Composition of Animal Feed to Study Body Weight Gain/Pound Feed
Ingested
Three different batches of animal feed were prepared. These batches
were referred to as (i) starter feed, (ii) grower feed, and (iii)
finisher feed. The compositions of the three batches of feed are
presented in Table II.
TABLE II % % % INGREDIENT STARTER GROWER FINISHER Corn yellow
56.429 62.115 62.017 Soybean_ML-48% 35.429 29.619 29.745 protein
Corn gluten ML-61% 0 0.467 0 protein Fat (3700 kcal/lb) 4.926 4.455
4.941 Methionine 88% 0.243 0.304 0.318 NaCl 0.321 0.278 0.289
Calcium Carbonate 0.678 0.680 0.692 Defluorinated 1.684 1.567 1.458
Phosphorus* Choline Cl-70% active 0.090 0.105 0.105 Trace Mineral
Premix 0.050 0.050 0.025 Bacitracin MD 0.050 0.050 0.050 Vitamin
Premix** 0.050 0.050 0.050 Coccidiostat*** 0.050 0.050 0 Liquid
marigold extract 0 0.202 0.310 *32% calcium - 18% phosphorus **16
common vitamins and essential nutrients ***AgriBio Corporation
(Athens, Georgia)
EXAMPLE 13
Preparation of Hemicellulase/Soybean Meal Feed Supplement
The procedure described in Example 3 was used in the production of
useful quantities of hemicellulase. The hemicellulase was recovered
as described in Example 8. Approximately 2.2 lbs of the resulting
hemicellulase paste was mixed with a small aliquot (8.8 lbs) of
soybean meal (48% protein) to achieve a concentration of
31.75.times.10.sup.6 units of enzyme activity per pound of
hemicellulase/soybean meal mixture. The mixture was allowed to air
dry in a large open pan under a fume hood. The dried mixture was
ground to a fine powder in a Waring blender and stored at 4.degree.
C. until used.
EXAMPLE 14
Preparation of Hemicellulase Containing Animal Feed
Three types of animal feed containing 2 lbs of the
hemicellulase/soybean meal mixture per ton of feed were prepared.
Two pounds of the hemicellulase/soybean meal mixture prepared
according to the method described in Example 12 were added to the
ingredients (excluding the yellow corn and soybean meal) comprising
each of the three types of animal feed prepared according to
Example 12. The hemicellulase/soybean meal mixture and the other
ingredients were mixed for one minute in a vertical agitator. This
mixture was then combined with predetermined amounts yellow corn
and soybean meal and mixed in the vertical agitator for 3.5
minutes. The resulting hemicellulase/feed mixture was formed into
pellets by processing the feed mixture in a California press mill
equipped to deliver moist steam (10% v/w). The feed mixture was
stamped into pellets which were cooled immediately in a vertical
forced-air cooling tower. The pellets were shaken to remove any
fine powder material, which was recycled to the pelleting chamber.
The starter feed pellets were crumbled by processing them through a
roller mill.
EXAMPLE 15
The Use of Hemicellulase as a Feed Additive
The effect of adding hemicellulase to animal feed containing
soybean meal as a factor of the average body weight gain of
chickens/pound of feed ingested was studied. One day old chicks
(Peterson X Arbor Acres strain) were selected for the study. The
birds were vaccinated against Marek's Disease and
Newcastle-Bronchitis. Twenty-five birds of each sex were caught at
random and weighed to establish an acceptable weight range. A range
of +/-5 grams of the mean was determined for each sex. Birds were
caught at random, screened for proper weight and randomly assigned
to a group consisting of 76 mixed sex broiler chicks (38 males and
38 females). In total, there were 7 groups of test and 7 groups of
control chickens.
Each group was housed in a separate pen. The temperature of the
pens was checked daily for the first two weeks of the study. The
house temperature and humidity was checked daily for the duration
of the study. Air exchange was enhanced by wall fans. The birds
were placed on built-up litter. Wet litter from previous studies
was removed and replaced by one inch of new litter. Artificial
lighting was provided continuously and all birds had ready access
to water.
All birds that died within the first 7 days of the study were
replaced with a bird of the same sex from the same shipment of
birds. No bird was replaced after 12:01 pm of Day 7 of the
study.
All chicks were placed on their respective diets on Day 0 of the
study. The chicks were fed crumbled pellets of starter feed on Days
0-21. On Days 22-39, the chicks were fed pellets of grower feed. On
Days 40-46, the chicks were fed pellets of finisher feed. The pens
were checked three times daily during the study. Birds that died
during the study were necropsied to determine the cause of death.
Birds which were unable to get food and water were culled. The
weight and day of removal of all dead and culled birds was
recorded. The chicks were weighed on Day 21 and Day 46 of the
study. Feed efficiency was determined by dividing the amount of
feed, in pounds, which the chicken consumed by the weight it
gained, again in pounds. The results of the study at Day 21 are
reported in Tables III, IV and V are illustrated in FIG. 5. Results
at Day 46 are reported in Tables VI, VII and VIII and illustrated
in FIG. 5. A statistical analysis of the results employing Duncan's
New Multiple Range Test is presented in Table IX.
TABLE III Pen Test/Control Avg Bird Wt (lbs) Feed/Gain 65 Test
1.184 1.366 66 Test 1.129 1.380 67 Test 1.205 1.358 68 Test 1.194
1.363 69 Test 1.216 1.368 70 Test 1.228 1.371 72 Test 1.290 1.364
64 Control 1.177 1.392 63 Control 1.130 1.388 53 Control 1.141
1.383 27 Control 1.062 1.419 30 Control 1.130 1.406 18 Control
1.103 1.385 71 Control 1.186 1.452
TABLE IV SUMMARY OF DATA AVG BODY WT AVG FEED/GAIN (lbs) DAY 21 DAY
21 Control 1.133 1.404 Test 1.206 1.367
TABLE V ANALYSIS OF VARIANCE DF* SS** MS*** F**** 21 DAY BODY
WEIGHT Treatment 1 0.0199 0.01909 13.30856 Replication 6 0.01635
0.00273 1.89955 Error 6 0.00861 0.00143 Total 13 0.04406 21 DAY
FEED/GAIN Treatment 1 0.00465 0.00465 12.14572 Replication 6
0.00173 0.00029 0.75371 Error 6 0.00230 0.00038 Total 13 0.00867
*Degrees of Freedom **Sum of Squares ***Standard Error of Mean
****Assessment of Significance
TABLE VI Pen Test/Control Avg Bird Wt (lbs) Feed/Gain 65 Test 4.970
1.902 66 Test 4.868 1.879 67 Test 4.672 1.936 68 Test 4.803 1.896
69 Test 4.908 1.925 70 Test 4.941 1.824 72 Test 4.820 1.859 64
Control 4.669 1.921 63 Control 4.695 1.933 53 Control 4.415 1.996
27 Control 4.205 1.924 30 Control 4.800 1.933 18 Control 4.586
1.901 71 Control 4.738 1.909
TABLE VII SUMMARY OF DATA AVG BODY WT AVG FEED/GAIN (lbs) DAY 21
DAY 21 Control 4.587 1.931 Test 4.854 1.889
TABLE VIII ANALYSIS OF VARIANCE DF* SS** MS*** F**** 46 DAY BODY
WEIGHT Trtmnt 1 0.25089 0.25089 16.11932 Repl 6 0.22965 0.03827
2.45906 Error 6 0.09339 0.01556 Total 13 0.57393 46 DAY FEED/GAIN
Trtmnt 1 0.00627 0.006272 20.78077 Repl 6 0.01290 0.002157 7.12865
Error 6 0.00181 0.00030 Total 13 0.02097 *Degrees of Freedom **Sum
of Squares ***Standard Error of Mean ****Assessment of
Significance
TABLE IX PROBABILITY LEVEL = 0.05 TREATMENT MEAN SIGNIFICANCE
Control-Avg Body Wt, 21 day 1.133 B Enzyme-Avg Body Wt, 21 day
1.206 A Control-Avg Feed/Gain, 21 day 1.404 A Enzyme-Avg Feed/Gain,
21 day 1.367 B Control-Avg Body Wt, 46 day 4.587 B Enzyme-Avg Body
Wt, 46 day 4.854 A Control-Avg Feed/Gain, 46 day 1.931 A Enzyme-Avg
Feed/Gain, 46 day 1.889 B
* * * * *