CA2105559A1 - Alkaline protease 3733, its production and use in cleaning contact lens - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The fermentation and isolation of a novel alkaline protease, protease 3733 is described. This enzyme has the properties of an elastase and a serine protease. This enzyme is produced by a newly discovered alkalophilic Bacillus sp. IAM 011105 bacterium. Protease 3733 is characterized by high activity in hydrolyzing denatured lysozyme and the use of this enzyme in cleaning contact lenses is described. The biochemical properties of the new enzyme are described and the activity of the enzyme on a variety of substrates under a variety of conditions is described and compared to other alkalineproteases.

Description

` PC~/~S 92/02597 ~O/US 29 . 1992 ALKALINE PROTEASE 3733, ITS PRODUCTION AND
USE IN CLEANING CONTACT LENS

BACRGROUND OF T~E INVENTION
FIELD OF THE INVEN~ION
This invention relates to a novel alkaline protease or elastase enzyme "protease 3733" having an activity capable of dissolving denatured lysozyme, a production method thereof, and the process of using protease 3733 in cleaning contact lenses DESCRIPTION OF RELATED ART

- Users of soft contact lenses must clean and disinfect their lenses for reasons of health and comfort In additlon to cleaning lenses with detergent, it is also necessary to remove protein deposit from the surface of the lenses The presently used method for removing deposited protein involves soaking the lenses in a protease (such as subtilisin Carlsberg) solution at room temperature, soaking in a protease in a thormal disinfectlng unlt, or soaking ln a ch-mical dislnfectant ~ h- d-naturetlon of prot-ln on the lens surfac- pr-sents a probl-m ln cl-anlng tho l-n~ p-clally wh-n th-rmal dlsinf-cting i~ us-d ~b- predomlnant proteln d-poslt-d on th- lens ls lysozym- secreted by the user's tear ducts ~he ~ ramoval of lysozym- from lens 1~ diffleult u~lng conv-ntlonal ¦ prot-a~-s, ~ueh as Jubtlllsln, e~p-clally when th- ly~ozyme ~`
, SUBSTITUTE SI~EET

PCTlUS 9~/!3259 7 RO/US 29 ;, 199, has been denatured by thermal disinfecting Tne present invention is a novel alkaline protease which overcomes the relative inefficiency of conventional proteases in removing denatured lysozyme from contact lenses SUMMARY OF THE INVENTION
The fermentation and isolation of a novel alkaline protease, also called an elastase, named "protease 3733" is described The enzyme was produced by fermentation of a newly isolated Bacillus sp strain IAM 011105 The structural and biochemical properties of protease 3733 are described and compared to other proteases This enzyme has an unusual degree of activity in hydrolyzing denatured lysosyme Denatured lysozyme is the prime contaminant affecting cortact lenses The new enzyme is also highly active on the insoluble fibrous protein elastln The ,~ use of protease 3733 to clean contact lenses is described BRIEF DESCRIPTION OF THE DRAWINGS
~ Fig 1a shows the effect of pH on activity of protease - 3733 in hydrolyzing azocasein Fig 1b shows the effect of pH on stability of protease 3733 at 4 C and at 23 C
- Flg 1c ~bow~ th- Sfect of t-mp-ratur- on activity of prot~Je 3733 ln hydrolyzlng azocasein Flg ld how~ th- fS-ct of 40 C at pH 8 and pH 10 on tabllity of prot-a-- 3733 Fig 1- show- the fS-ct of 50 C at pH 8 and pH 10 on tabillty oS prot-a-~ 3733 S~EST~ ~ U~ S~EET

PCTiUV ;,`~J3259 7 ROIUS 2 9 J UN l992 Fig. 2a sho~7s the effect of chelating agent EDTA and the serine protease inhibitor PMSF on activity of protease 3733.
Fig. 2b shows the effect of EDTA at pH 10 and 40 C on stability of protease 3733 Fig. 2c shows the effect of divalent cations at pH 10 and 50 C on stability of protease 3733.
Fig. 3 shows the comparative hydrolysis of denatured lysozyme by protease 3733, subtilisin Carlsberg, subtilisin aprE, and subtilisin BPN'.
Fig. 4 shows the comparative hydrolysis of denatured human milk lysozyme by protease 3733 and subtilisin Carlsberg with and without BME.
Fig. 5 shows the comparative hydrolysis of denatured human milk lysozyme by protease 3733 and subtilisin Carlsberg with and without BME.

Strain Isolation. Bacillus sp. IAM 011105 was isolated from the aeration basin of an activated sludge facility used to treat textile finishing waste. The waste temperature was 20 C and the pH was 10.4. A small volume ~0.1 ml) of waste slurry wa- added to 5.0 ml of 4glllter nutrient broth (Dlfco, Detrolt, Mich.) buffered at pH 10.5 with 0.05 M
3-cycloh-xyl~mlno-1-propan-sulfonic acld (CAPS; Slgma Chemical, St. Louls, MO). $his culture was incubated at 30 C for 10 day~. $he broth wa~ then streaked on solid medlum of above compositlon plus 1.5~ Nobl- agar and 10t sklm milk th-n incubat-d SUBSTI~U~E ~HEET

PCTlu~ 9~/0259 7 ROlU~ 2 9 JUN ~992 21~35~

at 30 C for 3 days Strain IAM 011105 was isolated as a single colony exhibiting hydrolysis of skim milk casein Strain IAM 011105 is a gram positive bacterium At pH
? 5 it grows as a straight rod 0 5 - 1 0um x 5 0 - 8 0 um At pH 9 0 it grows as long thin rods approximately 0 5 um x 5 0 to greater than 15 um Sporulation was not observed in any cultures Colonies on Tryptic Soy Agar pH 7 5 are cream colored, opaque, circular, and convex with an entire margin Colonies on Tryptic Soy Agar pH 9 0 are cream colored, opaque, irregular, and flat with an erose margin Biochemical characteristics of Bacillus sp IAM 011105 were determined using the Minltek Disc System (Becton Dickinson, Cockeysville, MD) The results are shown in Table 1 Bacillus sp IAM 011105 has been deposited under No 55142 with the American Type Culture Collection, Rockville, Maryland Tnese data indicate that strain IAM 011105 is a previously undescribed bacterial species of the genus Bacillu~
Enzvme Production and Purification This invention relates to the culturing of a bacterium of the order Eubacterlale~, family Bacillacea-, genu~ BacilluJ, and to collecting and purl~ylng prot~ 3733 from th- culture medium A f-rnbaeh fl~k eontalnlng S00 ml of 30 g/l tryptic soy broth buffer-d to pH 9 0 with 0 05M CAPS was inoculated with 1 0 ml froz-n stock of Bacillus Jp IAM 011105 The culture w~ ~hak-n ov-rnlght at 200 rpm at 30 C Thi- wa- uJed to inoeulat- a 14 l Chem~p f-rment-~ eontaining 10 l of the same SlJ8STITUTE SHEET

PCTJUS ~2/0259 ~S 2 9 JU,`~ l99 2 1 ~

media. Cells were grown for 48 hours at 30 C w~th an agitation of 1300 rpm with an airflow of 8 liters per minute. Cells were spearated from the culture broth via centrifugation in a Sorvall RC-5B centrifuge and the cells discarded.
Enzvme Durification. The clarified culture medium was concentrated with a YM-10 ultrafiltration membrane with a 10,000 dalton cutoff, obtained from Amicon, a division of W.R. Grace Co., Beverly, MA. 30 ml of concentrate was passed over a 100 ml SEPHADEX G-25 column equilibrated with 0.01M sodium acetate and 1.0mM CaCl, pH 5.5. SEPH~DEX is a trademarkfor gel separation media owned by Pharmacia, Piscataway, NJ .
The desalted material was applied to a Pharmacia FPLC NonoS
HR 10/10 column equilibrated with the same buffer. Protease 3733 was eluded from the column using a 90 ml linear 0 - 0.2M
NaCl gradient in the above buffer.
Tne reeulting preparation of protease 3733 was shown to be homogeneous via SDS-PAGE, IEF, and N-terminal sequencing.
Purified protease is stored at 4 C as a 1.0 mg/ml solution in column elution buffer with 50~ propylene glycol.
Proteln concentration wa~ determ~ned uslng the 8lo-Rad prot-ln a~-ay klt (Blo-Rad, Rockvllle C-ntre, NY).

Blochemical Prop-rti-r of Protea-e 3733. Flg-.
1a - how- s-veral bloch-mical prop-rtles of protease 3733.
Unl-ss indlcated, all data wer- determlned und-r the following condltlons. All tudies were done with protea~e 3733 prepared as abov- and stored as indicat-d. Enzymatlc acltvity wa~

S~8STITUT~ SHEET

PCT/US ,~/3~59 7 ROIUS 2 ~ ~`,1, 21~5~tj~

determined using azocasein (Sigma Chemical Co., St. Louis, MO) as the substrate. 0.5% azocasein was prepared in 0.05M tris (hydroxymethyl) aminomethane (Tris) buffer (Sigma Chemical Co., St. Louis, MO) plus 1.OmM CaCl adjusted to pH 8.0 with HCl.
100ul of an appropriate enzyme dilution was added to 900ul of 0.5% azocasein and incubated at 30 C for 10 minutes. The reaction was stopped by the addition of 300ul of 10~
trichloroacetic acid. ~he reaction mixture was centrifuged at 12,000 rpm for 2 minutes in an Eppendorf microfuge. 80Qul of supernatant fluid was withdrawn into a fresh tube containing 300ul of 0.5 ~ NaOH. The mixture was vortexed and the absorbance read at 420nm. One unit of activity is the amount of enzyme required to give an absorbance change of 1.0 in 1 minute.
In Figs. 1a - e, the Activity axis represents the ~ relative activity calculated by comparison of each point to the highest value, which is given a relative activity of 100.
Fig. la shows the effect on the activity of protease 3733 on azocasein found by varying the pH values from 4 - 12.
The data in Fig. 1a indicate that protease 3733 activity against azocas-in had a pH optimum of at least pH 11.
Flg. 1b show- th- ffect of pH on stabillty of prot-ase 3733. Th- nzym-, at concentration of 1mg/ml, was incubated at pH valu-~ from 4 - 12 at 4 C, da~h-d lln-; or at 23 C, ~olid llne, for 20 hr-. and the actlvlty was det-rmined. The data ln Flg. 1b. indicat- that prot-a-- 3733 wa- stable for 20 hrs. at pH 5 - 9 at 4 C and table for 20 hrs. at pH 7 -- SU8ST1T~'TE SHEET

PCi~ ,iJ2597 5~ ROIUS 29 i ~ S.

9 at 23 C.
Fig. lc shows the effect of temperature on activity of protease 3733. Enzymatic activity was determined at 10 - 70 C. The data in Fig. 1c indicate a temperature optimum of 50 C.
Fig. 1d shows the effect of 40 C on stability of protease 3733. The enzyme at a concentration of 1mg/ml was incubated for up to 120 min. at 40 C at a pH of 8, dashed line; or pH
of 10, solid line; and the enzymatic activity determined. The data in Fig. 1d show that protease 3733 is stable at 40 C at pH 8 or pH 10.
Fig. 1e shows the effect of 50 C on stability of protease 3733. The enzyme was incubated for up to 120 min. at 50 C
at a pH of 8, solid line; or pH of 10, dashed line; and the enzymatic activity determined. The data in Fig. 1d show that protease 3733 loses 50~ of its activity after 15 min. at 50 C at both pH 8 and pH 10.
Figs. 2a - c show the effects of various additives on the activity of protease 3733. Unless indicated otherwise, the conditions were the same as in Figs. la - e.
Fig. 2a shows the effeet of ~1) phenylmethylsulfonyl fluoride ~PMSF) and ~2) thyl-nediaminetetraac-tic acid ~EDTA) on proteas- 3733 actlvlty. The data in Flg. 2a ~how that PMSF
at 1.OmM inhibited the actlvity of protease 3733 whlle EDTA
at 5.0mM had no effect on enzyme activity.
Fig. 2b ~hows th- eff-ct of EDTA on the stability of SUBSTI I lJ ~ ~ SHEET

PCT!U~ 3 2 5 9 7 RO/US 2 9 ~ Y~
2i~5 protease 3733. The enzyme was incubated for up to 120 min.
at pH 10 and 40 C in the presence and absence of EDTA at a concentration of 2.OmM and the activity was determined.
Crosses indicated presence of EDTA points and triangles indicated control points lacking EDTA. The data in Fig. 2b indicate that EDTA drastically reduced the stability of protease 3733.
Fig. 2c shows the effect of divalent cations on stability of protease 3733. Chloride salts of a variety of cations were added to separate aliquots of enzyme solution which were held at pH 10 and 50 C for up to 120 min. At intervals indicated, samples were taken from each aliquot and the enzyme activity determined. All experimental salts were at a final concentration of 5.0mM. The experimental points on Fig. 2c are as follows:
triangle, control; cross, Ba ; open square, Ca ; closed square, Co ; cross in square, Mn ~; cross with hyphen, Zn~+; and diamond, Mg . The data in Fig. 2c indicat that Ca and, to a lesser extent, Mg increased thermostability. The data in Figs. 2a - c are typical for ~acillus alkaline proteases.
The specific activity of protease 3733 was examined on several substrate lncluding azocasein, N-succinyl-ala-ala-pro-ph- p-nltroanllide ~AAPF-pna), N-succlnyl-ala-ala-ala p-nltroanlllde ~AAA-pna), and lastln congo red, all obtalned ~rom Slgma Ch-mlcal Co., St. Louls, MO. Speclflc actlvities w-re compared to those obtain-d wlth subtilisin Carlsberg and subtlllsin aprE.
En2yme as-ays wlth AAPF-pna and AAA-pna as substrates w-re . .

SU8STI ~ UT~ ~HFET

PCTiUS ~2/325~ 7 RO/us ;2 v~ J iJN 199.
21i)~5~

performed as follows. 190mM AAPF-pna or AAA-pna was prepared in dimethylsulfoxide. Reaction mixtures contained 980ul of 50mM Tris-HCl (pH 8.0) ~ 1.OmM CaCl, 10ul of either AAPF-pna or AAA-pna, and 10ul of an appropriate enzyme dilution. The increase in absorbance at 41Onm, due to the release of p-nitroaniline, was followed continuously at 25C. One unit of activity is the amount of enzyme required to give an absorbance change of 1.0 in 1 minute.
Enzyme assays with elastin congo red were performed as follows. 1Omg of elastin congo red (Sigma Chemical Co., St.
Louis, MO) was weighed into 13x100mm test tubes. 900ul of 50mM
Tris-HCl (p~ 8.0) + 1.OmM CaCl was added followed by the addition of 100ul of an appropriate enzyme dilution. tubes were capped and incubated for 30 minutes at 37 C with shaking. the reaction was stopped by the addition of 1.0 ml of 0.7M XP04 (pH 5.5), the tubes centrifuged at 3000 rpm for 10 minutes, and the absorbance of the supernatant fluid read at 495nm. One unit of activity is the amount of enzyme required to give an absorbance change of 1.0 in 1 minute.
Table 2 gives the specific activlties of protease 3733 on the lndlcat-d substrates ln comparlson to those of Jubtill in Carl~b-rg and ~ubtlllsln aprE. Speclflc activltles are expressed a~ Unlt-/mg of enzym-. Tho data ln Table 2 lndlcate that the sp-clflc actlvity of protease 3733 on several of the substrates xamin-d ls slgnlflcantly dlff-rent from that of ~ubtlllsln Carlsb-rg and from that of subtlllsin aprE. The spociflc i SUBST~TUTE SHFET

PCTiU~ ?~)259 7 R0/US ~l~ .. lg9 2103~3 activity of protease 3733 is similar to that of the subtilisins on azocasein However, the specific activity of protease 3733 is higher than that of subtilisin aprE, and much lower than that of subtilisin Carlsberg on AAPF-pna The specific activity of protease 3733 is much higher than that of either subtilisin on AAA-pna The specific activity of protease 3733 is much higher than that of either subtilisin on Elastin congo red This indicates that protease 3733 is a different and distinct enzyme from either subtilisin Carlsberg or subtilisin aprE
Composition of Protease 3733 The amino acid compositions of protease 3733, subtilisin Carlsberg, and elastase Ya-B are compared in Table 3 Elastase Ya-B is an alkaline elastase isolated from Bacillus sp Ya-B as described in Biochim Biophys Acta, 1986, 833, pages 439-447 The enzymes were hydrolyzed and the resultant component amino acids analyzed Table 3 shows the number of amino acid residues which resulted from the hydrolysis of each enzyme The amino acid compositions of protease 3744, subtilisin Carlsberg, and elastase Ya-B differ significantly from each other and indicate that the~e are different enzymes Th- N-t-rminal s-quences of protease 3733, subtiliJin CarlJb-rg, and elastase Ya-~ were determined and ar- shown in th- ~-ctlon "S-qu-nc- Llstlng" aJ SEO SD NO l, SEQ ID NO 2, and SEQ ID NO 3, re~pectlvely Table 4 shows the N-terminal s-qu-nce of th--- nzym-s Star- ln Tabl- 4 lndlcat- amino acid- homologou- to protease 3733 Protease 3733 xhiblt~ SO~

SUBSTIT'~ITE ~HE~T

PCTIUS ~ 2 5 9 7 2~ RO/US 2` : 199 ~omology with subtilisin Carlsberg and 34% homology with elastase Ya-s in the N-terminal sequence of these enzymes This indicates that the three enzymes are distinctly different proteins Properties of Protease 3733 and Other Proteases Table 5 compares several biochemical properties of pronase 3733 with those of subtilisin Carlsberg and elastase Ya-B Unless indicated, these properties were determined as in Fig 1a - e Data for elastase Ya-B were obtained from the above publication The data in ~able 5 indicate that the pH optima were similar for each enzyme and all were serine proteases Protease 3733 had a much lower pI than elastase Ya-B and subtilisin Carlsberg and protease 3733 had a lower pI than serine proteases in general Protease 3733 exhlbited some degree of cross-reactivity with subtilisin Carlsberg antibodles whereas elastase Ya-B had none The ratio of elastin/casein degrading activity was highest for elastase Ya-B, with the ratlo for protease 3733 approximately nine times that of the ratlo for subtillsin Carlsberg In addition, an elastase from Bacillus subtilis was found to have a pH optimum of 9 0 and a molecular w-ight of 25,000 ~ subtllis lastas- ls an elastas- lsolated from B subtllls s descrlbed ln C-n-dl-n ~ourn-l of Mlcroblology, 1988, 34, p 855-859 Hvdrolv~l~ ot D-n-tur-d Lvsozvme Lysozyme hydrolysis a~ays were performed as follows A 1 0 mg/ml solution of chlcken eggwhlt- ly-ozym- (Slgma Ch-mlcal Co , St Louls, MO) w-- pr-par-d ln 50mM sodlum borate ~p~ 3 0) nd 1 0 ml ot thls solutlon w-s llquot-d lnto 13x100mm test tub-s The tub--. .

SUBSTITUTE S~.EET

ROIUS 29 JJ'N l992 2105~9 were capped and placed in a boiling water bath for 5 minutesresulting in denaturation of the lysozyme After allowing the tubes to cool 100 ul of an appropriate enzyme dilution was added and the reaction mixture was incubated at 37 C for 30 minutes The reaction was stopped by the addition of 300 ul of 104 trichloroacetic acid followed by centrifugation at 3000 rpm for 10 minutes The absorbance of soluble amino acids in the supernatant fluid was read at 380nm Assays using human milk lysozyme (Sigma Chemical Co St Louis MO) were performed using this same protocol ~ny changes in incubation temperature or incubation time are indicated where appropriate Fig 3 illustrates the enzymatic activity of protease 3733, subtilisin Carlsberg, subtilisin aprE, and subtilisin BPN on denatured eggwhite lysozyme Activity was determined by incubatlng 25ug of purified enzyme with denatured eggwhite lysozyme under the conditions descrlbed above The data show protease 3733 to be 5 - 6 fold more active at hydrolyzing denatured eggwhite lysozyme than the other proteolytic enzymes Fig 4 shows the effect of beta-mercaptoethanol (~ME) on enzymatlc activity of protease 3733 and subtillsin Carlsberg on d-n~tur-d ggwhlt- lyJozym- The data show a two-fold nh~ncem-nt o~ ly~ozym- hydrolysls by the additlon of 0 4~ ~ME
for both prot-a-- 3733 and subtll~sin Carlsberg This presumably wa- due to tbe reductlon of disulfid- bonds in lysozyme maklng th- prot-ln mor- Ju~ceptible to proteolysis Prot~ 3733 w~s cl-arly superlor to ~ubtlll~in C~rlsb-rg ln that protease SUBSTIT JTE St!EET

PCT/U~ , 9 7 RO/US 2 9 JUN l992 5~-~

3733 without addition of BME was 2 5 fold more active than subtilisin Carlsberg even when the activity of subtilisin Carlsberg was enhanced by addition of BME
Fig 5 shows the activity of protease 3733 and subtilisin Carlsberg on denatured human mil~ lysozyme with and without the addition of 0 4% BME Without the addition of BME, protease 3733 exhibitad a 4 - S fold higher activity on human milk lysozyme than did subtilisin Carlsberg As in the case of chicken eggwhite lysozyme as substrate, the addition of BME
resulted in a two-fold enhancement of enzymatic activity for both enzymes However, protease 3733 without addition of BME
was 1 5 - 2 fold more active on denatured human milk lysozyme than was subtilisin Carlsberg enhanced with BME
Protease 3733 ~reatment of Contact Lenses Protein-contaminated contact lenses may be cleaned by incubation in a cleansing preparation containing protease 3733 A suitable preparation is a sterile aqueous solution of 0 05 M sodium borate buffer pH 8 0 with protease 3733 at 1 mg/ml Contaminated contact lens are immersed in the preparation and held at room temperature for 30 min ~he lenses are rlns-d w-ll wlth st-rll- physlological sallne befor- use Proteln cont~mlnutlon 1J ff-ctiv-ly removed from contact lenses u~ing thl~ pr-p-r~tlon and mothod In an optlonal formulat~on for cleanlng contamlnated contact l-nses, 0 4~ BME may be lnclud-d ln proparatlon Sultable cleanslng proparatlons also may be formulat-d SU8STIT~J 1 E SHEET

PCT/US ,", J~ 7 -" 2i~5~ RO/US 2 9 ~ ~N 1992 in nonaqueous solvents, such as hexane, cyclohexane, ethanol, methanol, and dimethylsulfoxide. Such solutions may be buffered to insure a high level of enzymatic activity. The method of use is as for aqueous solutions.
The invention has been described in detail with particular reference to preferred embodiments thereof: however, it should be understood that variations and modifications can be made within the spirit and scope of the invention and the same should not be limited except as set forth in the appended claims.

SU8STITUTE ~EET

PCTiUv ~ 2 ~ 9 7 R0/US 29 J~N l9S2 210~5~9 Mobility Oxidase Catalase Spore Production Observed Acid Produced Fro~:
Anaerobic Dextrose Aerobic Dextrose +
Maltose Trehalose Sucrose Xylose Lactose Arabinose Mannitol +
Cellobiose Adonitol Inositol Raf~lno~e Rhamno~-Sorbltol Nannos-Galacto~-Phenylalanln- Deamina~e SUBST! T U I E S~!rT

PCTiUS 92/0259 7 R0/US 2 v , JN 19 16 2~5~

Ornithine Decarboxylase Lysine Decarboxylase Arqinine Decarboxylase Beta Galactosidase Nitrate Reduction Denitrlflcation Voges-Proskauer Citrate (as sole carbon source) Urease Hydrogen Sul~ide Indol .
Esculin Hydrolysis Starch Hydrolysis Casein Hydrolysis +

; Elastin Hydrolysis -SUBST!T~TE ~YFET

PCTiU~ ~, 2 / ~J 2 5 9 7 RO/~S 2 9 J UN ~992 SubstrateProtease 3733Subtilisin Subtilisin Carlsberg aprA

., Azocasein 14.4 16.7 16.2 AAPF-pna 393 4286 370 AAA-pna 22.8 10.0 1.8 Elast1n congo red 2.6 O.~ 0.2 '.:

SUBSTlTUT.r S~ ET

~C ,i ~ )2 5 9 7 2~55~9 - Table 3 Amino AcidProtease 3744SubtilisinElastase Carlsberg Ya-B

Asx 35 28 28 Glx 18 11 14 - Ser 22 32 26 Gly 40 35 37 ~lis 9 5 8 Arg 10 4 7 Thr 13 19 17 Ala 36 41 33 Pro 13 8 8 Tyr 13 13 4 - Val 23 31 21 Met 8 5 3 Cys O O O
Ile 13 10 10 Leu 19 16 11 Pb- 7 4 6 Ly~ 4 9 4 Trp 1 2 2 Total 2B4 273 239 SUBSTITUT~ SHEET

PCTllj~ ~2/02597 RO/US 2 9 JUN l99 ResidueSubtilisin Protease Elastase ~o. Carlsberg 3733 Ya-B

1 Ala Gln *Gln Gly ; Thr *Thr Xaa Val *Val Xaa Pro *Pro * Pro Tyr Trp * Trp Gly ~Gly * Gly s Ile *Ile * Ile , Pro *Pro Asn Leu Tyr Arg Ile *Ile Val Lys Tyr Gln Ala Ser Ala Asp *Asp Pro Lys Val Ile Val *Val Ala Gln Xaa Gln Ala Xaa Ser . Gln *Gln Arg -~` 20 Gly *Gly * Gly Pha Tyr Ph-I Ly~ Ph- Al~

SUBSTITUTE ~HEEr ,~, P~TIUS ~2/0259 7 9 JUN 19' Gly * Gly * Gly Ala Asn Ala Asn Gly * Gly Val * Val * Val Lys * Lys Arg Val * Val * Val Ala * Ala * Ala .

SUBST~TUTE C~LJF~T

~O/US 2 9 JUN 199 Property Protease Elastase Subtilisin 3733 Ya-B Carlsberg pH Optimum 11 0 11 7 10 0 Temperature Optimum C 50 60 60 Molecular Weight 27,000 25,000 28,000 pI 4 9 10 6 9 4 Serine Protease ~ +
N-terminus Gln Gly Ala X-reactivity With Subtilisin Carlsberg Antibody A~tivity Ratio El-stin/Cas-in 0 18 0 sa 0 02 SU8ST! T ' ~ ~!E~T
:

PCTiUS 9Z/32,9 7 R0/US 2 9 J ~1~ 1992 22 210~559 SEQVENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Fiske, Michael J.
Mlddlebrook, Susan M.
Steele, D. Bernie Barnitz, Joy T.
(ii) TITLE OF INVENTION: Alkaline Protease 3733, Its Production and Use In Cleaning Contact Lens (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) Addressee: Genencor International Inc.
(B) Street: 180 ~imball Way (C) City: South San Francisco (D) State: California ~E) Country: USA
(F~ ZIP: 94080 v) COMPUTER READABLE FORM:
(A) HEDIUM TYPE: Di5c (8) COHPUTER: IM~ PS12 (C) OPERATING SYSTEM: WS

SUBST~TI!T' ~FET

-~ PGTIU~J ~ 2597 - ~lO~g (D) SOFTWARE: Multimate (viii) ATTORNEY/AGENT INFORMATION
(A) NAME: Passe, James G.
. (B) REGISTRATION NUMBER: 29,966 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415)742-7500 (B) TELEFAX: (415)583-8269 (2) INFORMATION FOR SEQ ID NO:1:

. (i) SEQUENCE CHARACTERISTICS:
: (A) LENGTH: 28 amino acid residues (B) TYPE: amino acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (li) MOLECULE TYPE: protein (lil) HYPOTNE~ICAL: no r ~lv) ANTI-SENSE: no (v) FRAGMENT TYPE: -N-terminal fragment e i SUBSTIT~JTE SHEFT

PCT/ ~ 2 5 9 7 RO/US 2 9 j ~N l992 21~5~

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus sp.
(B) INDIVIDUAL/ISOLATE: IAM 011105, ATCC 55142 (C) CELL TYPE: unicellular bacterium (D) DEVELOPMENTAL STAGE: vegetative cells (vii) IMMEDIATE SOURCE: bacteria isolated, cultured; enzyme isolated, sequenced (ix) FEATURE
(A) NAME/KEY: N-terminal sequence of alkaline protease, protease 3733 .~(C) IDENTIFICATION METHOD: Biochemical assay (D) OTHER INFORMATION: active as alkaline protease especially active on denatured lysozyme and elastin, useful for cleaning contact lens (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:l:

Gln Thr Val Pro Trp Gly $1e Pro Tyr Ile Tyr Ser Asp Val Val X~a Xaa Gln Gly Tyr P~e Gly A~n Gly Val Lys Val Ala ' 20 25 ~3) INFORMATION FOR SEQ lD NO:2:

~, i SUBS~IT' IT~ JF~T

PCTIUS 92/0259, 25 21~ 9 SEQUENCE CHARACTERISTICS
(A) LENGTH 29 amino acid residues ( B ) TYPE amino acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE protein (iii) NYPOTHETICAL no (iv) ANTI-SENSE no (v) FRAGMENT TYPE -N-terminal f ragment (vi) ORIGINAL SOURCE:
(A) ORGANISM Bacillus subtilis (B) INDIVIDUAL/ISOLATE Var Carlsberg ~ (C) CELL TYPE unicellular organism ¦ ~vll) IMNEDIATE SOURCE enzyme obtalned comm-rcially, Z-qu nc-d (lx) FEATURE
tA) NAME/XEY: N-termlnal s-quence of al~allne protease, ~ubtlll~ln C-rlsb-rg ~C) IDENTIFICATION METHOD: Ploch-mlcal ~ay r f SUBSTITUTE SHEET

PCT/US 97/û2~9 7 RO/US 29JU,J19C

(D) OTHER INFORMATION: active as alkaline protease (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala (4) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acid residues (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: protein ~lll) HYPOTHETICAL: no :
(lv) ANTI-SENSE: no ~v) FRAGMENT TYP~: -N-t-rminal fragment SUBS T IT~,~F SH~ET

I~Q/US 29 JUN 199 27 2 l a5 ~ ~ 9 (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus sp.
(B) INDIVIDUAL/ISOLATE: Ya-B
(D) DEVELOPMENTAL STAGE: vegetative cells (G) CELL TYPE: unicellular bacterium (vii) IMMEDIATE SOURCE: enzyme isolated from culture, sequenced (ix) FEATURE
(A) NAME/KEY: N-terminal sequence of alkaline protease, elastase Ya-B
(C) ID~NTIFICATION METHOD: biocbemical assay (D) OTHER INFORMATION: active as alkaline protease (x) PUBLICATION INFORMATION
(C) JOURNAL: Biochim Biophys Acta (D) VOLUME: 833 (F) PAGES: 439-447 (G) DATE: 1986 :

- ~xl) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Gly Xaa Xaa Pro Trp Gly Il- A-n Arg Val Gln Ala Pro Ile Ala Gln S-r Arg Gly Phe Ala Gly Ala Gly Val Arg Val Ala , SUBSTITUT~ SH~ET

Claims (17)

We claim:

1. A Bacillus sp. alkaline protease "protease 3733" enzyme characterized by activity in hydrolyzing denatured lysozyme, as a serine protease, with a molecular weight of about 27,000 daltons based on SDS-PAGE analysis, an amino acid composition as set out in Table 3, a N-terminal sequence as set out in SEQ ID NO:1, a pH optimum of at least pH 11 0 with azocasein, a temperature optimum of about 50° C, a pI of about 4 9, and an activity ratio on elastin/casein of about 0.18.

2. A culture of the microorganism Bacillus sp. strain IAM 011105 , said culture being capable of producing the alkaline protease enzyme "protease 3733" in a recoverable quantity upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic substances.

3. A biologically pure culture of the microorganism Bacillus sp. strain IAM 011105, said culture being capable of producing the alkaline protease enzyme "protease 3733" in a recoverable quantity upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic substances.

4. A process for producing a novel alkaline protease "protease 3733", as recited in claim 1, which comprises the steps:
culturing a protease 3733-producing bacterium belonging to the genus of Bacillus in a culture medium, and collecting protease 3733 from the culture medium.

5. A process according to claim 4, wherein the protease 3733-producing bacterium is Bacillus sp. IAM 011105 strain.

6. A process according to claim 4, wherein the culture medium is cryptic key break buffered with CAPS buffer at pH
9Ø

7. A process according to claim 4, wherein culturing is conducted aerobically at about 30° C.

8. A process for cleaning protein contaminated contact lenses comprising the step:
soaking the contact lenses in a solution of alkaline protease 3733.

9. The process of claim 8 wherein the solution of alkaline protease 3733 is an aqueous solution.

10. A cleansing preparation for contact lens comprising alkaline protease 3733 in a buffered solution at an alkaline pH.

11. The cleansing preparation of claim 10 wherein the pH is about 7.0 to about 13.0

12. The cleaning preparation of claim 10 wherein the buffer is sodium borate.

13. The cleansing preparation of claim 10 wherein the pH is about 8Ø

14. The cleansing preparation of claim 10 further comprising beta-mercaptoethanol at a concentration adequate to enhance the activity of the alkaline protease 3733.

15. The cleansing preparation of claim 14 wherein the beta-mercaptoethanol is at a concentration of about 0.4%

16. The cleansing preparation of claim 10 wherein the solution of alkaline protease 3733 is a nonaqueous solvent.

17. The cleansing preparation of claim 16 wherein the nonaqueous solvent is chosen from the group consisting of hexane, cyclohexane, ethanol, methanol, and dimethylsulfoxide.