CA1219827A - Thermo-stable micro-organism - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention relates to a biologically pure culture of extremely thermophilic bacterium THERMUS AQUATICUS (Variety T-351).
The bacterium produces an enzyme CALDOLYSIN which exhibits pro-teolytic activity, particularly at temperatures of 65°C to 85°C and which is stable at temperatures up to 75°C at a pH range of 4 to 12.
The bacterium was isolated from a hot pool in New Zealand which was at 79° ? 4°C, low in sulphide and at pH 7.5 to 7.8. The cells were gram negative, non-motile, non-sporulating rods. The bacterium exhibits optimal activity at 70-80°C.

Description

~` ~Z~9827 This invention relates to a novel micro-organism capable of producing a thermostable extracellular proteolytic enzyme. This is a divisional of Canadian patent.application 357,832 filed on August 8, 1980.
It is recognised that there is a demand for thermo-stable proteolytic enzymes in the food, fermentation, animal feed and pharmaceutical industries which is not being entirely met. The inventors have succeeded in isolating a micro-organism from a hot pool in the thermal area of Rotorua, New Zealand, which organism is capable of producing a thermostable proteolytic enzym~
It i.s an object of this invention to provide starting material capable of producing a ther~ostable proteolytic enzyme and to provide a proteolytic enzyme which goes some way towards meeting the aforementioned demand or at least provides the public with a useful choice.
Accordingly the invention may be said broadly to consist in THER~.US AQUATIC~S (variety T-351), hereafter described more simply as THER~US T-351 (as herein defined) in a substantially biologically pure form_~
In another aspect the invention may be said broadly to consist in a process for isolating THERM~S T-351 which comprises isolating a sample of solution from a hot pool (at 79 + 4C) low in sulphide at pH 7.8 containing said micro-organism, preparing a culture from said sample and maintaining said culture at a temperature of 65C to 85C and a pH between 7.2 and 8.2 for a time sufficient to produce an ade~uate yield and isolating the THERM~-S T-351 from said culture during the ~ 2 ~

late log phase thereof.
In another aspect the invention may be said broadly to consis, in THERl~S T-351 whenever prepared by the foreg ~ng process.

The invention consists in the foregoing and also envisages constructions of which the following gives examples.
Definitions 1. THERMUS T-351 This micro-organism was isolated from Hot Pool No. 351 as marked on the map of "Whakarewarewa Hot Springs", 1:2000, 1st Edition by New Zealand Geological Survey (Bibliographic reference - Lloyd, E.F. 1974, Geology of Whakarewarewa hot springs, DSIR
information series No. 104 DSIR, Wellington, N.Z.). The hot pool was at (79 + 4C), was low in sulphide, at pH 7.5 to 7.8. It grew poorly below 60C. It was obligately aerobic. The -1~198;~7 cells were Gram negative, non-motile, non-sporulating rods. It is similar to THERMUS AQUATICUS (Brock et al., J. Bacteriol 98, 289-287; Degryse et al, Archives of ~.icrobioloqY 117, 18) but the inventors have noted a significant difference in cytochrome composition of between THERMUS-351 and THERMUS AQUATICUS. The product exhibits optimal activity at 70 to 80C and negligible activity below 40C. Oth~r properties of this microorganism are set out herein below and are also described in Hickey and Daniel, J. of Gen. Microbioloqy (1979), 114, 195-200.

2. CALDOLYSIN (3.4.21.14) . . .
This is a proteolytic enzyme produced by THER~US T-351 and isolated by the process described below. It is a protease having molecular weight 20,000 + 3,000. It has an isoelectric point of approximately 8.5 and its enzymatic activity is described below. This enzyme is stable at temperatures of 75C
and below, in the presence of divalent cations, particularly calcium ions. It is stable at pH values from 4 to 12 in the presence of calcium ions. Other properties and the method of preparation of this product are set out herein below.
EXAMPLE la. Isolation of THERMUS T-351 __ Samples were taken from the hot pool identified under the definition of THE~US T-351 herein above. Isolation was carried out by repeated sub-culturing at 75C of the organisms contained in a 1 ml sample from the hot pool, in 10 ml of half strength nutrient broth, pH 8.0: This was followed by growth in the medium described in example lb (i).

219~27 EXAMPLE lb. Cu Ztivation of T~ERMUS ~-351 (i~ Cultures were maintained on a.medium consisting of Allen's salts, (Jackson et al., Archiv. f~r Mikrobiol 88j 127-133), with 0.1~ w/v yeast extract (BBL) and 0.1~ w/v trypticase (BBL) in liquid culture at 7S C. The medium was adjusted to pH 8.2 prior to autoclaving. The final p~ was 7.5.
(ii) The organism was grown at 75C on.a similar medium . but with 0.3~ yeast extract and 0.3% trypticase.
500 ml batches were grown in 2 1 Erlenmeyer flasks in an orbital incubator, and either harvested for use, or used to inoculate.a.20 1 fermentor.
The organism grow well at 75C under the conditions (i) and (ii) and poorly below 60C. Cells were . harvested during late log phase (10-12 hours after inoculation) at an.absorbance of about 1.4 at 650 nm (about 2.5 x 107 cells ml 1) lc PreParation of subceZZuZar fractions:
Cell fractions.were prepared as.described by Daniel (Biochim.et Biophys Acta.216, 328-341) except that the sedimentation of.small particles was carried out at 250,000 g for 1 hour.
ld. ~easurement of o~ida~e.~tem activit~e~:
Oxygen uptake was measured polarographically using a Rank electrode tRank Brothers, Bottisham, Cambridge, England). The reaction mixture consisted.of 0.lM
KH2PO4/Na2HPO4 buffer pH 7.0, a.suitable.amount of membrane particle protein, 50 ~mol.of substrate (except in the case of.NADH.were 5 ~mol were used), in a final 0 volume of.2.5 ml.
Buffers were equilibrated at the desired temperature with sparged air for 30 minutes.
Particles were equilibrated in the electrode for 2 minutes prior to measurement of oxygen uptake. Rates were measured over the first minute.

`` 121g8Z7 le. spectrophotome~r?~:
Difference spectra were obtained at room temperature with a Cary model 17 recording spectrophotometer.
The concentration of individual cytochromes was determined from the dithionite-reduced minus oxidised difference spectra, and for cytochrome from the reduced minus reduced + CO difference spectra, using the following wavelength pairs ana extinction coefficients: c-type cytochrome, 553-540, EmM = 19 mM cm (Chance et a2 J. Bio.Chem. 217 439-451) total b-type cytochrome, 569-575 nm, ~mM = 17.5mM cm (Deeb & Hanger (J.Bio. Chem.
239, 1024-1031); cytochrome o-CO, 417-429, ~mM =
170mM cm (Daniel, _ iochim et B_ophys. Acta 216, 328-341); a-type cytochrome, 602-630 and 613-630 ~ M = 24mM cm 1 (Van Galder, (Biochim. et Biophys. Acta 118, 36-46).
~ashed membrane particles were able to oxidise other substrates including glutamate and malate / >0.05 ~mol 2 min l(mg protein) 7, and lactate, citrate, fumerate, glycerol and glucose / 0.005 - 0.02 ~mol 2 min (mg protein) 7. Except in the case of succinate and lactate, activities were enhanced by added supernatant and by NADH. Acetate, sucrose, mannitol and ethanol were not oxidised.
Both NADH and succinate oxidases had maximum activity at pH 7.0, and at a phosphate buffer molarity of O.lM, as determined at 75C.
The activities of NADH and succinate oxidases were determined after 2 minutes preincubation at temperatures between 40C and 95 C. Each was highest at 75 C in cell free extract and in large and small particles.
The NADH oxidase rate in respiratory particles was particularly temperature sensitive, the rates at 70C
and 80C being about half that at 75C. In all cases activity at 75 C was at least 10-fold greater than that at 40C.

At 75 C, apart from an initial partial loss of activity the respiratory chain in whole cells, cell free extracts and respiratory particles was relatively stable, but there was a substantial short term increase in succinate respiration of whole cells and endogenous respiration followed a similar pattern. At 90C this was found for whole cells and cell free extracts, but not washed respiratory particles. At 90C the succinate oxidase of whole cells and the NADH oxides of washed respiratory particles were substantially less stable than the oxidase activities of cell free extract.
These stabilities are appreciably greater than those reported for NADH oxadase from BaciZlus stearot~rmophiZus protoplasts (~isdom & Walker, (J. Bacteriol, 114 1336-1345).
The thermostability of the NADH oxidase activityof respiratory particles at 90C over a 15 minute period was unaffected by phosphate buffer concentration (0.01 M to 2.0 M), 1.0 M - MgSO4 or by 10 mg ml casein.
Stability was enhanced about 2-fold by 50~ (v/v) glycerol, 2.0 M - (NH4)2SO4, and 10 mg ml NADH. Rates were determined at 75 C.
Absorption peaks of a~ b, and c-type cytochromes in washed respiratory particles at 613 and 602, 559 and 555 nm respectively were recorded. The major a-type cytochrome had an absorption peak at 613 nm, which is --unusual: the troughs at 615 and g44 nm in the carbon monoxide spectra suggest that at least one of the a-type cytochromes is a terminal oxidase~ The trough at 561 nm and the peak at 417 nm indicate the presence of cytochrome o, and the trough at 550 nm suggests that there was some CO-reactive c-type cytochrome in the respiratory particles. The high speed supernatant contained at least two soluble c-type cytochromes since the ratio of the peaks at 420 and 426 nm varies somewhat -`` 1219~Z7 between preparations, and at.least one of these was C0-reactive.
b and c-type cytochromes in.the ~H~RMUS NH have been reported by.Pask-Hughes & Williams.~Scientific Progress at Oxford 62, 373-393) and a-605 and b and c-type cytochromes.in a T~RMUS A~UA~ICUS type organism by McFetters and Ulrich (J. Bacterial 110(2), 777-779).
Cytochrome concentrations / ~mol cytochrome (g.
pro-tein) 1 7 in respiratory.particles were a-602, 0.03; a-613, 0.06; total b-type, 0.89; o-0.21; total c-type 0.64: In the supernatant, c-type 0.79; CO-reactive.cytochrome.c, 0.02. These.concentrations are fairly typical of these found in other aeorobes.
All inhibitors tested produced.levels of inhibition within the range of.those found in other bacteria and there was no evidence that active sites were less exposed than in non-thermophiles. Terminal.oxidase inhibitors affected NADH and succinate oxidases equally, as did amytal. Rotenone had.more effect on.the NADH
oxidase, while Bathophenanthroline 2-heptyl-4-hydroxy-quinoline-N-oxide and antimycin A were all.more effective inhibitors of succinate oxidase.

lZ19~ 7 EXAMPLE 2a - T~o Ste~ CALDO~YSIN Purification Vol. 50 L Culture fluid Centrifugation 50 L Supernatant (1) CBZ-L-Phe-TETA-Sepharose*4B
~ . Affinity chromatography 2.5 L Absorbate (2) The culture fluid treated according to the flow scheme set up hereinabove co~es from Example lb. The centrifuga-tion is conducted in a continuous flow centrifuge at 27,000 g. The pH of the-Supernatant was adjusted to p~ 8 prior to its being passed through the affinity gel.

STEP Volume ~Protein7 Activity Specific Purification Yield (l ) (~g~ml) (PU/ml) Activity (fold) (~) (PU/mg) 1. Supernatant 20 24 .005 .25 1.0 100 2. Affinity Purified 1.1 14 . .079 5.76 23.2 73 The experimental data set out in Table 1 herein below.
EXA~:PLE 2b - MuZtistep CaZdol~s~n Purification.

The overall reaction scheme is set out in the following flow sheet.

* Trade Mark r ~
i"', g Vol. 50 L Culture fluid ¦ Centrif~gation 27,000 x g I continuous flow 50 L Supernatant (1) SPC25 ion exchange Retardate containing two minor proteases 49.5 L Eluate (2~

Millipore*Ultrafiltration 1,000 MW nominal cut-off ~ membrane filtrate ~
8 L Retentate (3) ¦ CBZ-L-Phe-TETA-Sepharose 4B
! affinity chromatography 2.5 L Adsorbate (4) \ lyophilised - Enzyme Powder Resuspended in Buffer Enzyme concentrate (5) /

1.5 L Purified Caldolysin (6) Gel chroma-tography on Sephadex G75*.
Trade Mark 1~9827 The majority of the details are set out on the accompanying flow sheet. However the ultrafiltration step concentrated the eluate (2) ten times. The retentate (3) subjected to affinity chromatography was adjusted to pH 8.
the absorbate 4 was eluted as a single peak with pH 2.7 O.lM acetic acid.containing lOmM Ca2+.
The enzyme concentrate (5) was eluted from the G75 gel column using an eluting.buffer at pH 8.1 and 10 mM Ca2+.
The data of the various steps in the reaction scheme are set out herein below in table 2.

~219~3Z7 . ~a 0 O N O . .

C4 ~ ~ ~ ~ N

Cl~ ~ ~ ~
O ~ ~ CO ~O
~:

¢ ~ N ~ ~D ~NC~ ~

_~
O
o . D7 0 ~ ~ ~ ~ ~ a~ 0 O ~ X O
E~ O ~ ~ ~ 1 ~ dP

¢1 ~ oo ~ ~ N ,~ U~
.a _~ U~
C~
U~ O
O o a~
U~ ~ ~~ O ~

Q~ N .,1 ~:

~ QJ
~ N ~rl a) ~o ~ u~
U ~U ~ ~0 a~ o L~
* t) ~ 0 *
0 ~ O ~~
C: Q,~,1 :1 11 0 P~ ~ N ~1 r1P, ~J P, ~ o U~
~n ~n ~n ~.¢ ~ ~ ~ S0, E~
.... , *
~ N r~l ~ U'l ~D *

~Z:l9~Z7 E~AMPLE 3: Properties of CALDOLYSIN
A. Phvsical (1) A molecular weight of 20,000 + 3,000 was determined by gel chromatography, SDS electrophoresis, and Gradipore* electrophoresis.
(2) Isoelectric point: 8.5 + 0.5.

(3) Response t~ inhibitors (Table 3) and enzymatic specificity indicate that CALDOLYSIN is a metal-chelator-sensitive lytic protease (see Morihara (1974): "Comparative Specificity of Microbial Proteases", Advances in Enzymoloqy 41, 179) with an active site serine residue.
B. Enzymatic CALDOLYSIN hydrolyses a range of high molecular weight protein substrates (Table 4) and some low molecular weight peptide substrates (Table 5). However, a number of common peptide analogues (protease substrates) are not hydrolysed (Table 5).
CALDOLYSIN lyses a broad range of Gram-negative bacteria, but few gram-positive microorganisms (Table 6).
C. Stability (1) Thermostability.
In the presence of 10 m~1 Ca , 100% activity is retained at temperatures of 75C and below for an extended period (no loss over 170 hours). Removal of Ca2+ markedly reduces thermostability. Half-life - data at temperatures between 75C and 95C are shown in Table 4, together with published data 8~7 on other thermophilic proteases.
(2) pH/Stabilitv CALDOLYSIN is stable ~in the presence of calcium ions at 20C) for protracted periods at pH values of 4.0 to 12Ø At pH 3.0 T / =
2 hours.
At high and low pH values (for example pH 4 and pH 10), incubation at elevated temperatures results in a marked reduction in stability.

INHIBITORS
Type of Action InhibitorConcentration % Inhibitor of Activity General EDTA12.5 mM 100%
Metal EDTAlO mM 70%
Chelator EDTAl mM 40%
EDTA. 0.13 mM

Cysteine - Iodoacetic acid 10 mM 60 Enzyme "2 mM
Inhibitor n0~ 25 mM

Cysteine - p-chloromercuri 5 mM
Enzyme benzoate2.5 mM
Inhibitor Zn-specifico-phenanthroline lO mM
Chelator " 1 mM

Ca-specific EGTA 10 mM 4s%
Chelator EGTA1 ~M 18%

Trypsin inhibitor l.0 Mg ml -Acid protease N-a-p-tosyl-L-lysine 3xlO mM
inhibitor Chloromethyl ketone HCI

Although the reasons are not fully understood and we do not wish to be bound by any one theory, the apparent inhibition of CA~bOlYSI~ by EDTA and EGTA is likely as the result of destabilisation caused by calcium removal, and the subsequent loss of enzyme activity as the result of autolysis.

HYDROLYSIS OF PROIEINS BY CALDOlYSIN
_ Substrate Rate of hydrolysis ~ of rate of (~280 min x 10 )casein hydrolysis . _ _ _ casein 3.33 100 ovalbumin 1.45 44 bovine serum albumin 1.33 40 haemoglobin O.9O 27 collagen 0.70 21 fibrin 0.65 18 . _ . . . _ Rate of hydrolysis ~ of rate of (440 min x 10 )azo-casein hydrolysis . _ _ azo-casein 2.75 100 azo-albumin 4.15 151 azo-collagen 0.87 32 _ (oA395 min x 10 ) . _ . .
elastin-congo red 0.25 approx. 7 HYDROLYSIS OF PEPTIDE AND PEPTIDE ANALOG~ES BY CAlDOlYSI~

_ _ Substrate ~ydrolysis Pond hydrolysed . _ Gly-gly - _ Gly-gly-gly Gly-gly-gly-gly - gly-gly Gly-gly-gly-gly-gly - gly-gly D-leu-gly L-leu-gly BOC-ala-try-met-asp-phe-NH2 CBZ-gly-phe-NH2 Acetyl-ala-ala-ala-OMe - ala-ala CBZ-gly-pro-gly-gly-pro-ala - gly-pro CBZ-gly-pro-leu-gly-pro + pro-leu Benzoyl-arginine ethyl ester CBZ-gly-p-nitro-phenyl ester Tosyl-arginine ethyl ester Benzoyl-arginine-p-nitroanilide Benzoyl-phe-val-arg-p-nitro-anilide ~ amide CBZ-gly-pro-arg-p-nitroanilide ~219~z7 TA~LE 6 LYSIS OF MICROORGANISMS AT 75 C ~Y CALDOLYSIN (20 ~q ml , 0 1 M
CH3COONa, pH 7.5) Microorganism ATCC Gram Complete Partial No Nwn`oers reaction a lysis lysis lysis . . _ .
Arthrobacter gZobiformis 8907 +
Arthro~aeter - + +
BaeiZZus eereus 9373 + +
BaeiZZus megaterium9 376 + +
BaciZZus eireuZons9374 + +
~icrococcus Zuteus - + +
Microeoeeus Zysodeikticus - + +
Saeeharomyees eerevisiae - + +
Sarcina Zutea 196 + +
SDoreformer (unidentified BaeiZZus) - + +
StaphyZococeus aureus 6571 + +
Streptomyces griseus 8136 + +
Agrobacterium tumefaeienslS955 - +
AZeaZigenes faeeiZis 8156 - +
AZeaZigenes viseoZaetis 8154 - +
Citrobaeter freundii - - +
Cytophaga johnsonae C4 - - +
Eseheriehia coZi B11303 - +
Escherichia coZi ~12 ~ ~ +
Eseheriehia eoZi ~12Hfr - - +
Eseheriehia eoZi w - _ +
Enterobaeter aerogenes - - +
Enterobacter cIoaeae - - +
XZebsieZZa pneumoniae 418 - +

Proteus vuZgaris 67 - +
Pseudomonas aerogenes - - +
SaZmoneZZa typhimurium - - +
Serratia mareeseens1377 - +
ShigeZZa fZexneri - - +
ShigeZZa sonnei - _ - +

a. Gram reactions quoted from Bergeys Manual of Determinative ~acteriology (1974), 8th edition, (Buchanan R.E. and Gibbons N.E., eds.) Williams &
Wilkins Ltd.

`-" 1219~27 ~W C~ U O
h O O O O O O O O O
~ o o In o u~ o 0 r~ co ~ ~ 0 0 a~ ~ o ~ U~
o E~ +l w ,1 -W . . - ~ ~
H r-l Ct) t~ ~r) O
~1 o co ~r o u~ ~ Jr 0 +
a ~4 A
~ ~ ~q T
r~ Cq ~ C~
~ ~ C~ ~ O O
W C) ~ ~ E:`t ~r) ,1 m ~: ~ ;~ :~ o h ~I P~ A, ~ ~ ~ ~ ~~ O
O O Ll ~ ~ ~ U~ ~U) C) C~ ~ ~ ~ ~ I I I I I
P: O~: ~ ~ ~ ~ ~ ~`
o :a A, 2 ;a E~
u~ ~ o ~ ~ o ~ ;~ ~ :a ~ ~ w 0 O
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-,~ O

~ ~ ~ 1`1 2 2 22 2 Z ~
o O P~ ~ ~ ~ `1 ~ .¢ H
O ~ O O O OO
S; 2 h~ ~ O ~1 ~ '`:1 . ~Z198Z7 (3) _tabilising Effect of Divalent Cations Table 8 shows influence of metal ions on the stability of CALDOlYSI~ at 85 C. ~12 ~g ml enzyme, pH 8.1 Tris acetic acid, I = 0.3 M 1 ).
CALDOlXSI~ was dialysed in the presence of 1.0 mM EDTA to remove any metal ion co-factors. -Standard metal ion solutions were added to aliquots of the "apoenzyme" to give lOmM concentration, after which the thermosta-bility of the enzyme was determined.

Metal ionHalf-life (minutes) Calcium est. 340 Zinc 144 Strontium 155 Magnesium 86 Cobalt 60 Barium 43 Copper 21 None est. 5-10

(4) Stability_to Denaturing Agents CA~DOLYSI~ has been found to be stable and active in the presence of a variety of denaturing agents as shown in Table 9.

~2198Z7 Stability of CALDOLYSI~ in the present of denaturing agents.

Denaturing agent Half life at 18 C Half life at 75 C

1% SDS >> 13 hours > 5 hours 8M Urea >> 67 hours53 minutes 0.8M Urea >> 13 hours148 minutes 6M Guanidine HCl >> 31 hours 59 minutes 1% Triton X100 - 60 minutes 1~ Tween*80 - 60 minutes .. _ . . . . _ . _

(5) Other CALDOL~SI~ is stable to eoneentration by lyophili-sation (freeze-drying) and rotary evaporation ~reduced pressure at 37 C) as shown in Table 10.

Method ofConcentration % Specifie ConcentrationFactor Aetivity Loss , _ Lyophilisation 7 7.2%
Rotary Evaporation 20 <5,0%
.. .. . . _ _ D. pH/Activity Relationships Optimum pH for aetivity on azocasein oeeurs-at 8.5 + 0.5.
(at 75 C). At pH 6.0 - pH 9.5, more than 80% of optimal aetivity is retained.
E. Tem~eratUre/ACtiVity Relationships Below 40 C enzyme activity is low (less than 6% of activity at 80 C). Aetivity rises almost linearly between 45 C and 80 C.
* Trade Mark -~` lZ19827 NB: Although % activity is low at normal temperatures (20 - g0C), sufficient activity for effective proteolysis can be obtained simply by using larger quantities of enzyme. However, its usefulness is clearly maximal at 65C - 85C.
EXAMPLE 4: ~E~M~S T-351 Growth and CAlDOLYSlN Production Optimum production of CALDOlYSI~ was achieved when ~HERMUS ~-351 was grown on peptone media containing Allen's salts at peptone concentrations of 0.6% to 1%, (Cell divi-sion time ~ 2 hours). Concentrations o peptones greater than 1% inhibited production. T~E~MVS ~-351 grew poorly on salts/casein or salts/albumin media, and excreted little protease. However, yields of extracellular protease could be increased significantly by addition of protein substrates to 0.6% peptone media. At 75C, optimum yield of CALDOlYSIN occurred within 18 hours (19% inoculum, aeration = media vol./min.).
Yield: 0.12 PU/ml culture medium : where 1 PU = lmg tyr released/min at 75 C; substrate = 0.5% casein.
EXAMPLE 5: Immobilisation of CALDOLYSI~ to Glass Beads CAlDOLYSIN was immobilised on non-porous glass beads by the silane glutaraldehyde-coupling method described by Stolzenbach & Kaplan / (1976). Methods in Enzymoloqy 44, 926_7. 10 g of glass beads (Corning glass, 100 mesh) was washed in an excess of 5% HNO3 at 100 C for 30 minutes. The acid-washed glass was filtered and rinsed, then added to a 10% aqueous solution of y-aminopropyl triethoxysilane (adjusted to pH 3.5 with HNO3). The suspension was incu-bated at 75C for approximately three hours to permit silanization to occur. After filtering, the silanized glass was added to a 20 ml volume of 5~ glutaraldehyde in 0.01 M, pH 7, phosphate buffer. This was reacted in vacuo for two hours ~t room temperature, and finally washed exhaust-ively with distilled water.
17 ml of a solution of CAlDOlYSIN (25 ~g ml ) of ~` ~Z19~27 - 2~ -known activity was added to the prepared ceramic substrate.
The suspension was stirred at room temperature for 18 hours to complete glutaraldehyde crosslinking The immobilised enzyme was subsequently filtered, washed with 100 ml H2O, 100 ml 1 M NaCl, and a further 500 ml H2O. The filtrate and washings were assayed by the Kunitz method.
The immobilised complex was assayed ~y a modification of the Kunitz method. 14 mg samples of the enzyme-bead complex were placed in reaction tubes, mixed with 2 ml of 0.5% casein substrate, and incubated at 75C with continual shaking. The proteolytic activities of the original enzyme solution, the immobilised preparation, and the washings (non-immobilised enzyme) were calculated (Table 11).

Activity of Glass-bead-immobilised CALDOLYSIN

. . _ Enzyme activity (PU) Total enzyme activity of original solution 25.4 Total enzyme activity not bound to glass beads 0.6 Total activity of ceramic-bound enzyme 0.2 Recovery of activity in immobilised state = 1%

. _ . .................... . _ . ..
It is concluded that CALDOlYSIN was either inactivated during the attempt to cross-link it to the silanized glass, or was bound in such an orientation that steric hindrance prevented access of the protein substrate to the catalytic site.
EXAMPLE 6: Immobilisation of CALDOLYSIN to Sepharose 4B
_ _ Sepharose 4B (Pharmacia) was activated with cyanogen bromide as described by Fujiwara & Tsuru / (1977) International Journal of Peptide and Protein Research, . . _ . _ 12~g~7 9, 18 7. During activation, the Sepharose suspension was maintained at 25c, and at pH 10 to 11 by dropwise addition of 4N NaOH. The activated gel was washed and stored at 4C.
15 ml of a CAlDOI,SYIN solution (25 llg ml 1 in 0.1 M CH3COONa, pH 7.2) was adjusted to pH 9.7 and added to 40 ml of.settled.activated Sepharose 4B. The mixture was incubated at 4C for 72 hours. Subsequently, the CAlDOlYSIlY-Sepharose complex was filtered and washed with distilled water. Assay results for the free enzyme, i.mmobilised enzyme and gel washings are presented in Table 12.
TA~LE 12 Activity of Sepharose 4B-immobilised CAlDOlYSIN

Enzyme activity (PU) Total activity of free enzyme solution 17.0 Total activity not bound to Sepharose0.7 Total activity of Sepharose-bound enzyme 12.0 Recovery of activi.ty in immobilised state = 73%

EXAl'lPLE 7: Immobilisation of CAlDOLYSIN to Carboxymethy cellulose The Curtius azide method, first described by Michael & Ewers ~ (1949) Makromolekular Chemie 3, 200 7 modified by Mitz & Summaria./ (1961) Nature 189, 576 7 and detailed by Crook.et aZ. / (1970) Methods in Enzymology 19, 963 7 and Lilly / (1976) Methods in Enzymol~ 44, 20 46 7 was used to immobilise CAlDOlYSI~ to CM-cellulose.
5 g of CM-cellulose*(Pharmacia) was treated with methanol in acid, hydrazine hydrochloride, and sodium nitrite in acid, as described in the papers cited above.
To the activated cellulose was added 77 ml of 25 CAlDO~YSIN (61.5 l~g ml in pH 9.2 buffer). The substrate-enzyme coupling reaction was accompanied by a decrease - * Trade Mark lZ19~ 7 in pH, which was read~usted to 8.7 by addition of saturated sodium borate solution during the 60 minute duration of reaction. The complex was subsequently washed with aliquots of distilled water, NaCl, acetic acid, and sodium bicarbonate solutions. The immobilised complex and all solutions were assayed as previously aescribed. Activity data are presented in Table 13.

Activity of CAlDOLYSIN immobilised to CM-cellulose Enzyme_activity (PU) Total activity of free enzyme solution 239 Total activity not bound to CM-cellulose (washings) 29 Total activity of CM-cellulose-immobilised CAlDOLYSI~ 66 Recovery of activity in immobilised state = 31%
_ _ . .
EXAMLDLE 8: Com~arative data for free and immobilised . CAlDOLlSIN
It has been shwon in examples 5 to 7 that the immobilisation of CALDOl~SI~ to various insoluble substrates occurs with considerable differences in the recovery of active immobilised enzyme (i.e. 1% for glass beads, 31% for CM-cellulose, and 73~ for Sepharose 4B).- This may be due to loss of activity by denaturation, or differences in inhibition due to the site of the enzyme-matrix covalent linkage.
The activity retained after immobilisation of CAlDOLYSIN to Sepharose (73%) was high when compared to other published data. In binding a range of proteases to Dowex*MWA-l anion exchange resin, Ohmiya et aZ. ~ ~1978 * Trade Mark . .. .

lZ~9t3Z~

Biotechnology and Bioengineering 20, 1 7, found activity yields ranging from 3% to 39%. Mason et aZ. / (1975) Biotechnology and Bioengeneering 17, 1019 7 obtained activity yields of 41.4~ and 57.7% on coupling ~. subt*Z*s neutral protease to glass by the azo- and glutaraldehyde methods, respectively.
A range of characteristics of the immobilised CA~DOLYSI~ preparations, including thermostabilities, pH activity profiles, and Michaelis-Menten kinetics, were compared with those of the free enzyme. Since the residual activity of the glass-bead immobilised enzyme was extremely low, no further study of this complex was carried out.
The thermostabilities of the immobilised CALDOLYSI~
preparations were determined at different temperatures and calcium concentrations. Volumes of immobilised enzyme were suspended in O.l M Tris acetic acid buffer, pH 8.1, containing known concentrations of calcium.
The suspensions were incubated at the desired temperature, and aliquots removed at intervals for assay after agitation of the suspension to ensure homogeneity.
Immobilised apoenzyme suspensions were obtained by eluting the insoluble complex (held in a Pharmacia K12 glass column) with 10 mm EDTA for several hours, and final washing with distilled water. (The term "apoenzyme"
is subject to the conditions discussed previously: it is possible that in the immobilised state, tightly bound calcium ions might not be removed by such treatment).
Thermostability data is presented in Table 1~.

lZl;9827 TABLE_14 A comparison of the thermostabilities of free and immobilised CAI,DOLYSIIY
.. _ .... _ .
Enzyme status Calcium Ca Half-life (minutes) at T C
status (mM) . . _ . _ _ Free Holo 10 360 60 28 Sepharose-bound Holo 10 1060 165 125 cM~cellulose-bound Holo 10 - 110 Free - Apo 0 - >6 Sepharose-~ound Apo 0 - 28 Free Holo 0 - approx.15 Sepharose-~ound Holo 0 - 64 The immobilisation of CA~DO~SIN on Sepharose results in an increase in thermostability of 3 to 4-fold over a number of different temperatures and conditions, while a thermostability increase of approximately 2-fold results from covalent linkage to CM-cellulose.
The decrease in stability of the holo-enzyme Sepharose complex when incubated in a calcium-free buffer suggests that the stabilisation by high calcium concentrations is as significant a factor in the immobilised state as -in the free enzyme, while the decreased stability ofthe Sepharose-immobilised enzyme after EDTA treatment (napoenzyme") indicates that immobilisation does not prevent the removal of at least some of the calcium-conferred stabilisation.
A sample of ~HERMUS T- 3 51 has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America under number ~219827 EXAMPLE 9: Stability of CAlDOLYSIN at varying concentrations of Ca~
The half-lives of CALDOLYSIN in the presence of different concentrations of calcium ions is presented in Table 15. The CAlDOLYSIN and ion solutions were prepared as in Example 3 (C).(33 above.

Ca+~ Concentration ~alf life mM min.
.. .. .. . .

o - <10 0.1 15 .

Claims (8)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. THERMUS T-351 in a substantially biologically pure form.

2. A process for isolating THERMUS T-351 in a substantially biologically pure form which comprises isolating a sample of solu-tion from a hot pool (at 79° ? 4°C) low in sulphide at pH 7.8 containing said micro-organism, preparing a culture from said sample and maintaining said culture at a temperature of 65°C to 85°C and a pH between 7.2 and 8.2 for a time sufficient to produce an adequate yield and isolating the THERMUS T-351 from said cul-ture during the late log phase thereof.

3. A process according to claim 2 wherein said hot pool is Hot Pool No. 351, Whakarewarewa Hot Springs, as identified by the New Zealand Geological Survey, 1st Edition, as herein defined.

4. A process according to claim 3 wherein said isolating step comprises repeated subculturing of said sample in a half strength nutrient broth at pH 8.0, the broth volume being substan-tially ten times the sample volume, at 75°C.

5. A process according to claim 4 wherein said culture comprises a liquid medium containing Allen's salts with 0.1 to 0.3 percent w/v yeast extract and 0.1 to 3 percent w/v trypticase at a pH of 7.5.

6. A process according to claim 5 wherein said pH is raised to 8.2 and the medium is autoclaved prior to innoculation with said culture.

7. A process according to claim 5 wherein said culture is harvested for use at the end of said culturing period.

8. A process according to claim 5 wherein said culture is used to inoculate a fermentation process.