CA1055416A - Production of thermostable lactase with bacillus organism - Google Patents

Production of thermostable lactase with bacillus organism

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Publication number
CA1055416A
CA1055416A CA240,972A CA240972A CA1055416A CA 1055416 A CA1055416 A CA 1055416A CA 240972 A CA240972 A CA 240972A CA 1055416 A CA1055416 A CA 1055416A
Authority
CA
Canada
Prior art keywords
lactase
organism
lactose
temperature
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
CA240,972A
Other languages
French (fr)
Inventor
Chin K. Lee
Margaret E. Long
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
RJ Reynolds Tobacco Co
Original Assignee
RJ Reynolds Tobacco Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by RJ Reynolds Tobacco Co filed Critical RJ Reynolds Tobacco Co
Application granted granted Critical
Publication of CA1055416A publication Critical patent/CA1055416A/en
Expired legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C21/00—Whey; Whey preparations
    • A23C21/02—Whey; Whey preparations containing, or treated with, microorganisms or enzymes
    • A23C21/023—Lactose hydrolysing enzymes, e.g. lactase, B-galactosidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
    • C12Y305/02006—Beta-lactamase (3.5.2.6)

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

ABSTRACT OF THE DISCLOSURE
A thermostable lactase is obtained by cultivation of a member of the genus Bacilius in a suitable nutrient medium by ctlng such a ~3r which ~xh5bit~ opt~um growth at ~ to~p-selecting such a member which exhibits optimum growth at a temp- c~ or th- coll-fr~a ~nzy~n3 ar~ us~ful for t~ hydrolysls of erature of at least about 45°C. The resulting lactase-containing cells or the cell-free enzyme are useful for the hydrolysis of lactose in a variety of substrate media.

Description

. oss4~6 This invention relates to a process for produc-ing lactase from a heat-toleran~ organism belonging to the genus Bacillus.
There has been considerable interest in recent years in developing methods for reducing lactose level3 in milk and milk-derived products. This interest has been .
heightened by recent evidence that a large percentage of the human population suffers from a lactase deficiency which is either an inherited trait or a result of the aging pro-cess. Such lactase deficiencies lead to intestinal dis-order3 when dietary levels of lactose are high. Moreover, a similar laatose intolerance has also been observed in certain domestic animals.
The hydrolysis of lactose3 in milk and milk-derived products to produce glucose and galactose is an ,~
attractive goal not only becau3e it would solve the lactose intolerance problem but it would also increase the sweet-ness of the products and reduce so-called sandy textures in certain milk-derived products caused by laotose crystal-lization. Those working in the field have long appreciatedthe desirability of effecting this bydrolysis by the use Qf lactase. In spite of khe fact that lacta~e occurs rather ;~ .
widely in nature and is produced by many microorganisms, the use o~ lactase in the commercial production of milk and milk-derived produat6 has been very limited. One
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~L0559~6 reason for the limited commercial use of lactase is that many of the common lactases such as those derived from yeast exhibit optimum enzyme activity at temperatures which are also conducive to bacterial growth. Accordingly, there has been increasing interest in finding a lactase having a high degree of hea~ stability. Such a heat stable lactase would permit lactose hydrolysis to be carried out under conditions that are unfavorable for growth for certain bacteria which are commonly present in milk or milk-deriv~d products. One such lactase derived from Streptomyces coelico~lor has recently been described in United States Patent No. 3,816,259. It is not clear at this time, how-ever, whether lactase from S. coelicolor can be used with impunity because certain members of that species have been reported to produce antibiotics.
The production of lactase by members of the genus Bacillus has been previously reported. P.J. Anema has reported in Biochim. Biophys. Acta 89 (3), 495-502 (1964) the isolation of lactase from B. subtllis. Lactase from B. me~atexium was described by S.R. Rohlfing and I.P. Crawford in J. Bacteriology 92 (4), 1258-9 (1966~.
~. :
Neither o these organisms, however, is regarded as heat-tolerant and the lactase produced by them must generally be used at temperatures below about 50C. in order to re-tain u~eful enzyme ackivity for extended periods of time.
In accordance with the present invention thereis provided a proceas for produci~g a lactase having im-~ proved thermal stability which comprises cultivating an ; organism belonging to the genus Bacillus in a nutrient medium containing lactose and recovering the lacta~e thereby
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1~554~6 produced, the organism being one which exhibits optimum growth at a temperature of at least about 45C.
A preferred organism for use in accordance with this invention has been isolated from a soil sample.
Culturing the organism in a suitable nu~rient medium con-taining lactose produces the desired lactase intracellular-ly. Characterization of this culture has been carried out and it has been identified as falling within the species Bacillus coagulans according to the classification given in Bergey's Manual of Determinative Bacteriology, 7th Edition. This organism is included in the culture collec-tion of the U.S.D.A. Northern Regional Research Laboratory under the designation NRRL Ei-8100. The taxonomic properties of this strain are shown in Table 1. ~ -TABLE I
A. Morphological characteristics 1. Vegetative RDds: Less than 0.9 micron in diameter varying in length generally up to 5.0 - 6.0 microns. Some fila-ments. Not in chains. Gram positive, staining uniformly.
Motile. :~
2. Sporangia: Generally not swollen but occasional swollen sporangia may be found.
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3. Spores: 0.9 by 1.2 to 1.5 microns, ellip-~oidal, subterminal to terminal.
B. Cultural Characteristics 1. Gelatin agar streak plate -- No hydrolysis.
2. Agar colonies -- Opaque, small, round. Not distinctive.
3. Agar slants -- Scant to moderate growth. ~lat, ~mooth, opaque.
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4. Glucose agar slant -- Growth heavier than on nutrient agar. Smooth white.
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5. Glucose asparagine agar slants -- No growth at 24 hours. Moderate growth at 48 hours.
6. Proteose peptone acid agar slants -- Good growth, better than on nutrient agar.
7. Soybean agar slants -- Growth moderate, slightly heavier than on nutrient agar.
`' 8. Stock culture agar slants -- Growth scant at 24 hours, as good as nu~rient agar at 48 hours.
9. Broth -- Growth poor after 24 hours.
10. Sodium chloride broth -- No growth in 7% sodium chloride.
}5 ll. Milk agar streak plate -- No hydrolysis.
; 12. Potato -- Scant, dry, wrinkled.
C. Physi~logical Characteristics l. Using peptone as the nitrogen source, the organism produced acid but no gas from glucose, lactose, ~arab:inose, xylose, mannitol and maltose. Neutral reaction from sucrose and~glycerol.
2. The pH of glucose broth is 5.0 or less in seven days.
3. Citrates not utilize~.
4. Tomàto yeast milk curdled in 24 hours at 45C.
25~ 5. Nltrites not produced from nitrates~
6. Voges-Proskauer tes~ i9 negative. Ph o~ Voges-Proskauer broth is 4.2.
7. Hydrolysis of starch - Positive.
8. Catalase - Po~itive.
30 ~ 9. No growth in nitrate medium under anaerobic condi-tions. Growth in glucose broth under anaerobic con-ditions produces a pH less than 5.2 in seven days.
:-~, ~ , ~ 10. Aerobic, facultatively anaerobic.
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11. Minimum temperature for growth is 25C. Maximum temperature or growth is 60C. Optimum growth occurs at 45-50C.
- As indicated unfler the physiological characteristics listea in Table l, the Bacillus organism disclosed herein 5~
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exhibits optimum growth at temperatures of about 45-50C~
and is, therefore, regarded as a heat-tolerant organism in comparison with other Bacilli which display optimum growth at about 37C. A heat-tolerant Bacillus organism is de-fined herein as one which exhibits optimum growth at temp-eratures of about 45C. and above.
E X A M P L E
____ The pH optimum of the lactase produced by this organism was determined by assaying whole cells in the presence of a-nitrophenyl-~-galactoside (O~PG) as the sub-strate. The procedure used was essentially that of J. Lederberg as described in J. Bacteriology 60, 381 tl950).
Cells were first treated with toluene, a phosphate buffer was used and the assay temperature was 37C. hactase activity was observed in the range o~ about pH 4.5 to 8.0 with optimum activity occurring at about pH 6Ø
Stabili~y of the lactase enzyme produced by B. coagulans was evaluated by using ONPG as the substrate in a modification of the Lederberg procedure. For this evaluation washed cells were suspended in 0.05M pho~phate buffer at pH 7.0 in the presence o~ the ONPG substrate.
The suspenslon was then maintained at 60C. for ~our days with ~amples being periodically withdrawn for determining the lactase activity remaining. The temperature for this ~i 25 stability test was selected as approximating the temperature levels used in low temperature pasteurization. Results of `
the test are sbown in Table 2.

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Lactase Activity as Per Cent Time in Hours of Original Activity 100 ~

The lactase produced by Bacillus coagulans exhibits useful enzyme activity up to about 70C. Optimum ~-activity appears to occur at temperatures of 60 to 65C. -.

The particular temperature selected for effecting lactose - : :
. . . .hydrolysis by the use of this enzyme will depend somewhat :

on the substrate medium involved. Generally speaking, however, temperatures between about 45 and 65C. are pre- :

!~ ferred.
.
~3;~ A typical medium for cultivating Bacillus co-agulans to produce lactase is as follows:
Proteose peptone 1.0% ~ :
: 20 Yeast extract 1.0%
Potassium dihydrogen phosphate 0.8%
: Lactose (Rterilized separately) 2.0 pH 6.0 Cultureis are incubated on a rotary shaker for 48 hours at :25 45C. At the end of the incubation period toluene is added to the broth ~0.5% on a volume per volume basis) and the mixture is agitated for 30 minutes. Cell~ are then recover-ed by floccuLation techniques described in United States Patent No. 3,821,086 and the floaculated cell aggregate thereby obtained is dried at 55C. Lacta~e activity of khe '~

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~055416 dried aggregate particles is 38.5 units per gram where a -unit is defined as the quantity of enzyme necessary to pro duce one micromole of dextrose per minu~e under the assay conditions. The assay method used for this determination is that of Weetall et al~, as published in Biotechnology and Bioengineering 16, 295 (1974).
The effectiveness of the lactase produced by Bacillus coa~ulans was demonstrated by the hydrolysis of lactose in a sweet whey feed stock. The organism was cultivated and the cells were recovered as described above. ~ -The dried aggregate particles wsre sieved and 5 grams of the 16-20 mesh portion were hydrated in a S0 per cent lactose solution buffered at pH 7.0 with a 0.05M phosphate -buffer. The hydrated particles were then packed into a small glass column that was maintained at a ~emperature of 60C~ Through this packed column was passed continuously an aqueous solution containing 70 grams of a commercial ,~ dried sweet whey powder per liter, the solution being buffered with 0.05M phosphate at p~I 7Ø The feed solukion ~ 20 contained approximately 5 per cent by weight lactose based -~ on the lactose content of the sweet whey and 100 milligrams .
per liter of methyl p-hydroxybenzoate was added as a pre-~~ ~ servative. Flow rate through the column was maintained at '~ 375 milliliters per day and the degree of lactose hydrolysis was monitored daily by routine analysis. Initial degree of lactose hydrolysis was found to be 90 per cent. After 3 weeks of continuous operation the degree of lactose hydrolysis had decreased to 80 per cent.
; It will be appreciated tha~ the lactasq produced in accordance with this lnvention mav be used for either batch ... . .
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~L0554~1L6 or continuous treatment of lactose substrate media. More- :
over, the lactase may be utilized by direct use of the cells or it may be used in the form of cell-free enzyme by apply-ing techniques known to those skilled in the art.

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Claims (7)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A process for producing a lactase having improved thermal stability which comprises cultivating an organism belonging to the genus Bacillus in a nutrient medium con-taining lactose and recovering the lactase thereby produced, the organism being one which exhibits Optimum growth at a temperature of at least about 45°C.
2. The process of claim 1, wherein the organism is cul-tivated in the nutrient medium at a temperature of at least 40°C.
3. A lactase prepared by the process of claim 1 which exhibits activity in the range of about pH 4.5 to 8.0 with optimum activity at a temperature of 60 to 65°C. and a pH
of about 6.0, the thermal stability of said lactase being such that at least 80 percent of the original activity is retained after approximately 21 hours of incubation at 60°C.
as determined by assaying lactase activity associated with washed cells of said Bacillus organism suspended in a 0.05 M
phosphate buffer at pH 7.0 in the presence of .sigma.-nitrophenyl-.beta.-galactoside.
4. A process for hydrolyzing lactose to product glucose and galactose which comprises contacting lactose with a lac-tase produced according to claim 1, under lactose-hydrolyzing conditions and recovering a product containing glucose and galactose.
5. A process according to claim 4, in which lactose is contacted with the lactase at a pH between 4.5 and 8.0 and at a temperature between about 45 and 65°C.
6. A process according to claim 5, in which the organism is Bacillus coagulans.
7. A process according to claim 6, wherein the organism is NRRL B-8100.
CA240,972A 1974-12-04 1975-12-03 Production of thermostable lactase with bacillus organism Expired CA1055416A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US52932974A 1974-12-04 1974-12-04

Publications (1)

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CA1055416A true CA1055416A (en) 1979-05-29

Family

ID=24109461

Family Applications (1)

Application Number Title Priority Date Filing Date
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Country Status (8)

Country Link
BE (1) BE836246A (en)
CA (1) CA1055416A (en)
CH (1) CH611647A5 (en)
DE (1) DE2554407A1 (en)
DK (1) DK143714C (en)
FR (1) FR2293490A1 (en)
GB (1) GB1493542A (en)
NL (1) NL7514110A (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS53148591A (en) 1977-05-31 1978-12-25 Daiwa Kasei Kk Production of novel lactase
NZ190603A (en) * 1978-06-07 1982-03-23 Nat Res Dev Heat-stable -galactosidase derived from bacillus stearothermophilus hydrolysis of lactose
DK146325C (en) * 1979-01-10 1984-02-20 Novo Industri As BETA-GALACTOSIDASE, METHOD OF PREPARING IT AND USING THEREOF
US6428786B1 (en) 1993-09-28 2002-08-06 Mcneil-Ppc, Inc. Composition and method for lactose hydrolysis
US6057139A (en) 1995-06-29 2000-05-02 Mcneil-Ppc, Inc. Preblend of microcrystalline cellulose and lactase for making tablets

Also Published As

Publication number Publication date
GB1493542A (en) 1977-11-30
FR2293490A1 (en) 1976-07-02
DK545175A (en) 1976-06-05
DK143714C (en) 1982-03-22
FR2293490B1 (en) 1979-08-10
CH611647A5 (en) 1979-06-15
BE836246A (en) 1976-06-03
DE2554407A1 (en) 1976-06-10
NL7514110A (en) 1976-06-09
DK143714B (en) 1981-09-28

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