AU1730399A - Enzyme stabilazation by block-copolymers - Google Patents

Description

rUU.U'~ Regut-aiof 3 2(2) AUSTRALIAk Patents Act 1990

ORIGINAL

COMPLETE

SPECIFICATION

STANDAR .D PATENT Application Number: Lodged: EN1ZYME STABILZATION BY BLOCK- CO)POLYMERS Invention Title: The following statement is a full description of this invention, including the best method of performing it known to us ENZfME- STAL ZT7 7ON SY_ 3LOCK1-CODOLYXSP',=R Technical ;'ielci The field Of the invention is u'ne scaoiliv-ation oz enzymes by means of- a ncn-ionic novathe-olvol block-copolymer surfaccant.

Backcrround art Enzym'es generally are formul ated into acrueous-nasea 1 iouid enzvmatic comositions designed for a particular process. These- liquid e nz -m a Lic compositions, however,X have historically been plagued with.1 oroblems such as chemical- instability wHi ch 'cana r e SUl in the -LOSS f enzvmatic activitvy tuartcula-y unon storac. Tis.

*cri ti cal Droblem o f loss of enzvmnatic acti-vity upon storage has Darticulariv affected the iouiQ azeceroenu industr-v.

is t is not uncommon no nave industrial oroauctS, such as-liquid enzymatic compositions, stored in'-warenouses in .variouis climates around the world where the p:lroduct- is subjected to a -temoperazcure that may range from. freez ing to above 50 0 C for extended periods. After stora-ae at temoerature extremes ranging from 01C to 501C 'for many months, most liTud enzymatic compnositions lose from 20 to 100 Dercent of -thir enzymatic activity due to enzytaie instability.

Various attemots have been made to stabilize enzymfes -containea in liouL-d enzymatic compositions- A.ttmots to -increase the stability of liqu~id enzymatic compositions -using formulations containinig alcohols, c-vcerols, di-lkylglvcolethers, and mixtures of these and other compounds have na, onlyv marginal success, even i n moderate storage temperature ranares- In Munk, U.S Patent No- 4,801,544,, a system of -ethylene glycol and ethoxylated linear alcohol nonionic -7 sur :act~rnr With hvdrocarori soiv-enc was ut±1---s.

stabiliizer and the encapsulat-on of enzYMeas in micelles wihi te olen/sr~~tfl mxtre was escribed. The waercnten~t of the c0111052.iiof was ket at: less than..

pecnand enzyme stab.'litiy was check'ed at 350, 7Q0 and The st abiliJzat ion o f an acaueous enzyme. Lpreparation using certain esters has been described by Shaer in U.S.

P atent No. 4,548,727. The ester used as a stabilizer has the formula, RCOOR', where R is an alkvI oc f-rom one to three' carbons or hydrogen, and RI is an alk- l of from one to six carbons. The escer _s Dresent in tne aau-eous enzyme preparation in an amount from 0.1 t~o abouL 2.5?k by weight.

The enzym inrdent: that- is eao-,e according to rhe *patentee is a commercial enzym nreparation sold in a dry *powder, solution or slurry -form containing from about 2 percent. to about 80 percent or actV enymes and a carrier such as sodium or-calcium sulfate, sodium chloride, glycerol, non-ionic surfactants or mixtures thereof as the remaining 20 percent- to 98 percent.

Letton et U-S. Patent: No. 41318,818 describes a stabilizing system for aqueous enzyme compositions where I...the stabilizing system comprises calcium ions and a low molecular weigh carbox-ili*c acid or its salt- Th-2 DHof the stabilizing system is frmabout e.5 to about !Q.

Guilbert et US_ Patent No. 4,243,1543 teaches the Sstabilization of liquid proteolytic enzyme-Containing detergent compositions. The detergent compositions are stabilized by adding an antioxidant and a hvdrobhilicpolyol to the compositulon while stabilizing the p-A of -the comp)osition.

Weber, tES. Patent No. 4,169,817 teaches a- icuid -aaigofpsiincnann stabilized enzymeS. The_ comaniig icus'Lan aeu o lutiioncnanfg rmlnt 4 4 by weight of solids and including detergent builders.

surface active agents, an enzyme system derived from Bacillus subtilus and an enzyme stabilizing agent. The stabilizing agents comprise highly water soluble sodium or otassium salts and/or water soluble hydroxy alcohols and enable the solution to be stored for extended periods without deactivation of the enzymes.

Dorrit et al., European Patent No. 0 352 244 A2 describes stabilized liquid detergent compositions using an amphoteric surfactant.- Kaminsky et al., U S. Patent No. 4,305,837 describes stabilized aqueous enzyme compositions containing a stabilizing system of calcium ions and a low molecular weight carboxylic acid or salt and a low molecular weight Salcohol. This stabilized enzyme is used'in a detergent Scomposition. The composition may include non-ionic surfactants having the formula RA;CH CH20) nH-where R is a hydrophobic moiety, A-is based on a group carrying a reactive hydrogen atom and n represents the average number of-ethylene oxide moieties. R typically contains from about 8 to about 22 carbon atoms but can be formed by the.

condensation of Dropylene oxide with a lower molecular t* weight compound whereas n usually varies from about 2 to about 24. The low molecular weight alcohol employed may be either a monohydric alcohol containing from.- to 3 carbon S atoms or a polyol containing from 2 to about 6 carbon atoms i "and from 2 to about 6 hydroxy groups. Kaminsky et al. note that the -olyols can provide improved enzyme stability and include propylene glycol, ethylene glycol and glycerine.

Tai, U.S. Patent No. 4,404,115 describes an aqueous enzymatic liquid cleaning composition which contains as an enzyme stabilizer, an alkali metal pentaborate, optionally with an alkali metal. sulfite and/or a polyol. The polyol contains 2-6 hydroxy groups and includes materials such as

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0 p- 4 Uf) 0 Us W1 V. 0~ 4) 0H 44 w' 0 4 3 N 4)i "3 0 .0 .3.1 C I 0 W1 0- 4 0 N, 0 In o 1) r-i (1 0 4 Id U)'0 3.1) a) I -1l 3.1 rd43 4 .41 03. OWN, S-' a scab~iiic- amtount: ofannoi o-ehrplo block-conolvmr~ su-1fanz.

elevatedi temme ratnurs the surfaczanz is sel ected to have a cloud creat:er znan suc- Lemu-aLures.

in one emdzOditr :1 t, tno n -ioi ovte-OA block--coooaivme'- is~acar a olovlvee vo ether all_-block, blcIn~c eteric -block or hecericheteic block cooo.lvmr whre the alkvlene units have fromn 2 to about I- carnon. atomis and csnaciallv those surrfactants wn icn cornta'4n nvoc-ronhon2.c ano hvcroohilic blocks w~here eachi block: is based on at least oxvethylene grouns or ox-voronvlene croups or ixtrsof thase arouos.

Teinventcion also comnor~ses a composition or- matt:er based on the foregoing en-m and surf actant- It is to be understood that both the foregoing general OdescriDtiofl and the -Folowina detailed descriotion are exemular- and e-xo!anatorv anda are 4-tended to nrovide =urther. exolanation of the 1ivCn:lon as claimned.

Best Mode for Ca-rrna OUt Tflvenrion *The oresent ZflveftIof Is directed to a method -for elevated temneratures or- mv wat-er, by COMnn~i ing the enz-yme 1:ith a non-ionic nocl eher-nolvo-lok Taliin ano enzmie andirist- tos oratiity, eiter a Jraptdi oe the latseveral years. As is well-known, enyes can be acid, alkalin.e or neutral, depending upon the uH range in wnicn- thev are active. Lipase alone or an with the followina enzvmes can DC used. All of these types of enzymes are contemplated to be useful in connection with the invention disclosed 'nerein.

Maniy as -'Lmasd cening C-fomlations av addition to thei-r assinC~nO w~lia d1U. eteracrits, U en~taS an nmnO5t1O5 nae !so shown -L III LY t a rnu7.ner of di-f flt c -c-al andu industri~al areas in whic'a a wid -aarit oL enzyvme ClaSSeS are flOw use__- Proteases are a well- -nown. class or- enzymfes :frecTuefl 2 utilized in a wide va-i tV o ad nusztr-Jal applacations where thlev act to hvflro.Lyze D-0a-d honds innoC.Sand orcteinaceous suosutrazes' PoteaSes are used to 'help t reov ~ot nse sans sucfl as niood or eac s~aifls.

Licula enzyim t-c comaoi;tionS contain alaln roueases have also shown to beuseful as dispesanlts of bacterial films and algal and 'f.nqal mazs in coolina tower waters and Meawr--n f Iu id c -a"'met hays.

Proteases canb-D~cnaacterizeQ as acd, neutral, or alkal ine Daroteases de 1- i n c unon tnhe Dt: raniei hc 2.they are active. The acid poroceases include tne microbial

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renets, rennin (c1hyosi:n) pepsin, and funa-ci oroteases. Trie neut~ral proteasas ilncluQe EtrvDsi, oalfl-, bromelaificfl-, and bactieria neutral Drotease. The alkaliae nroteases inld suntzixsin and related DroteaSeS. Commercial lia-aid.enzymtatic co-moositiofls con- *-tamning proteases; are available under the names Renni lase®D, IIDTNI' (Pancreatic Trvosin NOVO) "PEM" (Proteolvtic Enzyme Mxtue),Neutrasel), lklcalase®D, Esperasev, and Savinase- Al ~which are all sunn 1 l-ied by Novo Nordisk 3ioindustrialS, Tnc.

of Danbury, CT. Aother commercial urotease is available under the name FroeltCsupplied by Solvay Enzyrme-- Products- 'Amylases, another class of enzymes, have also been utilized- inmany industrial and commercial orocesses wnzc~'~V ct:cocazalvze or acce 1 craze the hvdralvsis OZf st-ch- As c-11lass arw-ases include c(:t-imv ase,3 atv ase, artwlog! ucosi dase (glucoamv-i se) ,*uncja amyl1ase, andn ull1u lana s e Commercial liauid e-'zvn-ttc coraOOS!ttons conzaining a-icvlases are a-vailab-!-- un-r th name s BA-N Termamvl6, MG, F'unqamv!S and Promozvm, wnichn are sunoliie bv Novo Nordisk, and Diazyzme L-200-_ a no-uct of Sol'vav Enzyme- Products.

Other comtrciafllv valuable enzyme classes are those~ which affrect the fVQ-rolyS-s of fiber. These class s, inrclude cellulases, hemicall~oe Ietnse n glucanases. Cellulases are enzymes that darace cellulose, a linear alucose polvia- occur--na tic-e walls of Dlants. -Heuicelluloses are involved in. tne hydrblvst's o f hemicallulose wHich, -1i ke cellulose. 'Is a- nolysaccharide fou-nd in ulants. The pectroases aree- u novdi the degr-adat--on of neccin,. a carbohvdrate whose main, component is a sugar acid. -lucanases are enzym es ilavolveQ in the hydrolvsi-s of B6-aluc ans w4h'-,ich are also simiar to ce 1 lu~lose jij that thlar inear nolvmers or-f alucose.

Coll-ctively, cellulases include endocell1ul ase exocellulase, exocello-biohydrolase, and cellobiase and for *the Duroose of the nresant invention will also include heriicellulase. Commercal licuid enzymatic comnos itn containing cellulases are available under the names CelluclastO and Ncvozv,.T0188 which are both supplied by Novo Nordisk Hemnicellulases that may be used include Che xvlanases.

PULPZYMs oroduct, available from Novo Nordisk, and ECOPULPt product, from A:,k6 Biotechnolo-v, are two examules or commercially available liquid enzymatic comoositions containing xvl,,anase-based enzymes.

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As a class, Ineicel-lulases _'nclude InicfllJIulasC rx zure anda aaiaczo,'-'.ar-naase. Commercia 1 liouid enzvm&Etc comfloslti~n con .lrc; nem cellulase are available as PULPZYM- 'fromtn Nova, ECOPULP f-om ~ika E iotcbnoloay and- N ovo z v m250 and -amaaase-, which are both oroduc s of Nova Nor_-di S'K The necci-nases that miav be used comorise endo-oo-lvaalact--ur onase, exooolv-cral1actu-onase, encopectate Ivase (transeli minase) exonectate !vase (transelimi nase) and andonect-i lvasa (transeliminase). Commercial .li-iid exizvmatic camD o s ition- s cantarin-ina nectinases are available under the names Pectinex- UltLra SP and Pectinex-*, both sunniijed by Nova Nor1disk.

The 0 -zlucanases that may be used comorise lichenous, laminar-inase, and exoglucanase. Commercial liquid en- ZVIR~atic coMposit!-ions containi-ng f-glucanases are available under the names Novozv.N234_ Cereflnv, BAN, Fizi.and I ~Ceremiixt, all orf which are supplied by Nova Nordisk.

in- addition to lipases, and Dhosff-olipases may also be! used. Linases and Dhospholinases are esterase enzymes w -lich hydrolyze f~sand o-- 1 -s by attacking the ester bonds In Cnese coMoou-nas- i-;iases act on tr2.glycerides, while- Dhosnholinases act on onosonoliopids. Tn the industrial sector, liopases arid ohosoholipases represent the camrnercially available esterases. Novo Nordisk markets two -ioruid lioase DreDarations under the names Resinase- A and Resinase- A 2X_ Commercial licoad enzymatic compositions containing liases are available- For exaimole, such camnositions are available under the trade names Linolase 100, Greasex SOL, Palatase-A, ?alatase-M', and Linozyme- which are all supplied by Nova Nordisk- Another commerialv valuable class of enzymes are thie isomerases which catalyze conversion reactionis between

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r1 II) 0~C] S -4 0 CI) I 004 4 u C: 0 I a1) (CI bi Si to Cd 4.1 0~ 0 to I Si U~ 9*b 9 aineS can he use aI obaned by~O cus fee-S a series or 5 ufctats can -he characz er sr 2CS -'a wattlfl1 and foam-: -jowe C can Deccs.Y er..tm-oved ne O~ttt7O: 0_ E:-e it

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aenera1-vdernas h--asi~ on 0= zne surfaczanzs.-' The ln-itial camonen- aoes not ,ava to be Ihv,.dropflQoi- hvaroflaODICitY rom one~ or- t'rwo c~ b'ocs The c~'nature o-fn cocnn U, omtOf D~-Te cenerally Ceter-i-ls the class caio- Oi~t. f rtesat:h. Tri starting m-arialS in s or coicaonents incude Mon'o Ydric alcohols such a tnn.,eh olDocn! buzanol and the likce as wel as cnr-cmaterials sucn. as clvcol.

Thie various classes of r .efer-- surfac'anzs, tC.L -ror practfl.ce of the Oreseant, 1:nV e nt1 h-i-n-ave -been 6escrlibed bv Schnmolk-a in qNnT-l.CSrctns. 'aczta- Science Series Vol- 2,ShcMJ. t ac D-r n. ew York, 1967, Chanter 10 w-ichn is incorporated hereln.D refernce- The -rst~ ano6 simmoleszt Is tha -nn eac.block is hom~ogeflec s wnl_4c.- i-,s t-o sayv a s ingl e a 1kylPI oXide is used in the mono-mer feed during eacn step in tne -arenaration- Sucna mater-ials are re: -re to as 11bo'surfactants. The,- next classes are termecdl~~n~i and heter1CO!O ;n nwhic'n one ~rino n oeue(~.

ei ther the hydroohobe or IsdCnlC corroosed of a s~iCl all-ylene oxiQe the- oz-er is a mixture or tw-,o or more such materiats, one or0Z nra n h aea

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1i1 that o-f the homogeneous block portion or-- tne molecule in the nreuaration of such materials, the h etero portion Or the molecule will be totally random. The properties or these non-ionics wi-'ll be entirely distinct from those or the pure block surfactants. T-he other subclass is that in which both steps inl the preparat ion of the hydrophobe and hydrophile involve the addition of mixtures of alkylene oxides and is defined as a heteric-heteric block copolymer.

The block polymer surfactant is typified by a monofunctioniA start--ng material such as a monohydric-alcohol, acid, mercantan, secoindary-.amine or N-substituted amides.

Such maeiascani generally-he illustrated by the following formwulawhere i is 'the startlnga rari1al molecule as described before. The A port'o r ,hycroonobe comprising an alkylene oxide unit in. r1cn. at ,lest: one hydrogen has been reoiaced by an alkyl group',or an arr-yl group, and w. is the degree of polymerization' wnich' is usually greater than about 6. The B moiety is-an aaueousisolubilizing croupsuch as oxyethvlene with n again being the degree o4f' polymrerization. The value of x is the functionality oZf1- Thus, where 1 is a monorunctional alcohol or amine, x is where I is a_ Dolyfaunctional starting material such as a diol propylene glycol)-x is 2 as is the case with the Pluronicv surfEactants. Where I is a tetrafunctional starting material such as ethylenediamine, x will be 4 as is the case with Tetronicv surfactants- Preferred surfactants of this type are the polyoxypropylenepolyoxyethylene block copolymers.

Multifunctional starting materials may also be employed to prepare the homogeneous block su'rfactants.

in the block-heteric and heteric-block materials either A or B will b~i a mixture of oxides with the t..

-12remainina block being a homogeneous block. One block will be- the hydrophobe and the ot-her1 the hydroDphile. E~tner of the two polvtieric units will serve as the solubiizing unit but tne characteristics will differ depending on which i.s emnoloved. Multifunctional starting macerials can also be employed in materials of this type.

The heteric-heteric block coooly_.ers are nrenarec essentially the same way as discussed previously with the major difference being_ that the monomer feed f or the alkylene oxide in each step is composed of a mixture of two or more materials. The blocks will therefore be random couolymers of the monomer. reed witnh the solubilitv characteristics deter..ined by the relative ratios of notentiallv water soluble ana war-r insoluble materials.

The average molecular weight of the polyoxyalkylene g lvcol ether block copolvmLers utilized according to the present invention is rrom about 500 to about 30,000 esvecially from about, 800 to about 25,000 and prefferably from about 1,000 to about 12,000. The weight ratio of hydrophobe to hydrooh-Ile will also vary from about 0.4:1 to 2-5:1, especiall1y froim about 0 to about 1.8:1 anda Dreferablv from about 0.8:1 to about 1.2:1.

'J ~In an especially pre-ferred emnbodiment, these surfactants. nave thle general f~r'ula: Rx(cH 2 CH 2 0) H where the hydrophobe of the block copolymer has an average molecular weiaht of from about 500 to about 2,500, especially from about 1,000 to about 2,000 and preferably from about 1,200 to about 1,500 and where R is usually a typical surfactant hydrophobic group but may al so be a polyether such as a polyoxy-propylene group or a mixture ofE polyoxypropylene and polyoxyethylene groups- In the above Xoml xis-either oxy en or nitrogen or another functionality capable ofL linking the nolyoxyethylene chain to the !7vdroo'nobe. In mos- cases. n, tace average number oz oxvethylene up-its -in the hydropnhil group, must be greater than about 5 or about 6 to impart sufficient water solubility to make tnhe materials useful.

The rDo7LVOXVa~lkvfln glvCol ethers are the prfered nonr-ionic Dolvether-nolvol block-cono±Yvmer surfactants.

However, other non-ionic block-coolv)mer surfaczants useful is the invention can be mod -d block- corolymers using the following as starting materials: alcohols, fatty acids, alkylphenol derivatives, glvcerol and its Qerivatives, fatty amaines, )-1,L-sor-Ditan derivatives, castor ci'l and derivatives, and glycol derivatives.

Cloud no-int is one of the most distinlct characteristics -for most non-ionic surfactants and depends on the number of" oxyetbylene, =ymopyiene, and/or oxybutylene groups reacted in the formation of the surf actant block coolyvmers of: the uresent invention.

Cloud voint is also affected by other comoonents in solution, the concentration oz- surfactants, and the solvents, i ry, in the system. Cloti Doint has been Qefined as the sudden onset oz turbidity o" a non-ionic surfactant solution on raising the temperature. When the non-ionic surfactant is dissolved. in water, it is theorized that an increase or temueratur.e will increase the activity of the wtrmolecules, which cause the dehydration of ether oxygens in the oolyoyxyethylene group in the non-ionic f sur-factant. Molecules with greater percentages of oxyethylene groups have a greater capacity for hydration, and so have a higher cloud ooint. This is important in the stabilization of enzymes in solution, since the long-term stability of the enzyme is evaluated at a temperature of SOOC. If the cloud Doint of a non-ionic surfactant is less than 500C, when the solution reaches that temperature, the enzyme will hydrate while the surfactant has coalesced and becomes less water soluble.

Cloud point has also been described as that characteristic of the non-ionic surfactants in which they exhibit an inverse temperature-solubility relationship in water, which is to say that as the temperature of the solution is increased, the solubility of the surfactant decreases. This phenomenon has been attributed to a disruption of specific interactions such as hydrogen bonding between the water and the polyoxyethylene units in the molecule. The temperature at which comnonents of the polyoxyethylene surfactant begin to precipitate from solution has also been defined as the "cloud Doint." In Sgeneral, the cloud point of the given family of surfaccants will increase with the average number of oxyethylene groups.

The cloud point of the non-ionic polyether-polyol surfactant block copolymers and especially the polyoxyalkylene glycol ether surfactant polymers of the present invention is greater than the temoerature at which i the enzyme or enzyme system degrades and may be anywhere from about 0 C to about 110 0 C, especially from about to about 100 0 C and preferably from about 20°C to about i 950C. These cloud points are for a 1 weight solution of A the surfactant in water.

I. Although the inventors do not want to be limited by a any theory, it is believed that the non-ionic surfactants of the present invention contribute to the stability of the enzyme by increasing the viscosity of the water in the formulation. Generally, high viscosity will .ead to poor transport to the Ca rich zones in enzymes such as protease, or slower ion transfer. This also helDs to keeD i, the matrix of the enzyme intact, although in some of the 4-f r-;S 7 a 15 cases described according to the'present invention, the higher viscosity may not be necessary for stability.

Chelating agents generally deactivate enzymes, decreasing the molecular compactness of the enzyme and causing deformation of the enzyme. Non-ionic surfactants are not influenced by the electrostatic effect, by the charged groups on the enzyme, and so do not impact on the special structure of the enzyme.

A suitable polyoxyalkylene glycol ether blockcopolymer that may be used according to the present invention contains a hydrophobe based on a hydrocarbon moiety of an aliphatic monohydric alcohol containing from 1 to about 8 carbon atoms, where the hydrocarbon moiety has attached thereto through an ether oxygen linkage, a heteric mixed chain of oxyethylene and 1,2-oxypropylene groups.

The weight ratio of oxyethylene groups to 1,2-oxypropylene groups in the hydrophobe is from about 5:95 to about 15:85 and the average molecular weight of the hydrophobe is from about 1,000 to about 2,000. A hydrophile is attached to the mixed chain and is based on oxyethylene groups. The weight ratio of hydrophile to hydrophobe is anywhere from about 0.8:1 to about 1.2:1. This polyoxyalkylene glycol ether is further defined by Steele, Junior, et al., U.S.

Patent No. 3,078,315 which is incorporated herein by reference.

One of the preferred polyoxyalkylene glycol ethers is Tergitol XD produced according to the method of Steele, Jr., et al. U.S. Patent No. 3,078,315 and available from Union Carbide. This is a non-ionic block copolymer having a cloud point of about 760C as a 1% solution in water and a molecular weight of about 3120 based on its hydroxyl number.

Other non-ionic polyoxyalkylene glycol ether blockcooolvmers can be employed such as those manufactured by the B-zS=F Wvandottle Coroorat-ion including Pluronics and TetrOnlict tvnes. PluronicO arid TecronicO nolyol surractants varv fro-a mobile luasto fLiak-able solids and those with. hig'h ethylene oxice contents exhibit no solution cloud noint even at 100 0 C. Other similar non-ionic Dolyoxyalkylene glycol ether block-copolymer surfactanus can be em-aloyed sucn as those manufactured by Dow Chemical Comoanv and Witco Chemical Corporation.

The PluronicO surfactants that may also be emploved according to the present invention are orepared by.

synthesizing a hydrophobe of desired molecular weight by the controlled addition or nrooviene oxide to the two hdroxv 1 grOups of popoylene glycol. Ethylene oxide is *then added to both ends of the nyavrconobe to fEorm oxyethylene chains that constitute from about 10 wt. to about 80 wt.A of the final mrolecule. The average molecular weight of the PluronicO surfactant is -from about 1,100 to about 12,G00 and the HLB (hydroooe lipophobe balance) is Pluronict P-105 employed according to the uresent invention has an averaae molecular weightL of about 6,500, a melting point of about 35 0 C, a cloud point of about: 91 0 C and an H-IB fabout 12-18. TetronicO surfactants thatmyalob employed according to thle invention are tetra-functional Itblock copolymers derived fr-the sequential addition o'r: propylene oxide and then ethylene oxide to ethylene- diamine. The average molecular weight of these surfEactants is from about 1,650 to about 30,000 and have an HLB of from about 1-7 to about 1-8-23 and greater than about 24.

Tetronicv 1304 emoloyed according to the invention has an average molecular weightof about 10,500, a melting point of about 59 0 C, a cloud point greater than about 100 0 C and an 'ILB greater than about 24.

17 17 The enzyme and surfaccant may also be used in combination with an organic solvent compatible with the enzyme and which will also act as a solvent for the nonionic polyether-polyol block-copolymer surfactant. The solvent preferably is hydrophilic such as a polyol or a mixture of polyols where the polyol has from 2 to about 6 j carbon atoms and from 2 to about 6 hydroxyl groups and includes materials such as 1,2-propane diol, ethylene glycol, erythritan, glycerol, sorbitol, mannitol, glucose, fructose, lactose, and the like.

The stabilized enzyme composition according to the present invention, therefore may contain an enzyme in an amount from about 2 to about 95 parts by weight, especially from about 5 co about 90 parts by weight and preferably from about 10 to about 80 parts by weight, water in an amount from about 1 to about 90 parts by weight and especially from about 2 to about 85 parts by weight and preferably from about 5 to about 80 parts by weight, a ''solvent from about 0 to about 70 Darts by weight and especially from about 2 to about 60 parts by weight and preferably from about 3 to about 55 parts by weight and the i'.i non-ionic polyether-polyol block-copolymer surfactant in an amount from about 0.2 to about 40 parts by'weight and especially from about 0.8 to about 30 parts by weight and preferably from about 1 to about 25 parts by weight.

The following examples are illustrative_ Example 1.

The composition listed below was made from Pulpzyme HB, an aqueous enzyme suspension, commercially available from Novo Nordisk Bioindustrials, Inc. which is a xylanase preparation with a bacterial origin. Tergitol XD, as described above was also employed. The glycerol used is a 96% pure material where the impurity is water. A higher purity glycerol may also be employed. The glycerol acts as

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a solvenz for Tehgirol XD, which is a_ solid at room h temneracure. VissOitv of tfle tormulat2on is 2,200 cus measured, by using a Brookfield vjrscosI n'Ite_ model number IRT, at 30 r-or, spindle number 4 at room temnerature .(20 0

C

The -formulation dissolves easilyv in water. Enzyme M activi:ty, TU rocr was measured accor-c to tne met-hod of sailey, M4.j. ec al., j. Biotech. 23, 257-270, 1992.

Tisi method entails.a five-minute -incubation of the xqvianase enzyme (suitably diluted. in pH 5.3 citrate buffer) wi~th a birchwood xylan substrate. After incubation, the released sugars are determined by a 5 minute reaction with the original DINS reagent of: Sumnwrer (1921) Absorbance is measured at 540 nm against a reagent blank comuriseQ orf substrate, DNS reagent and bu~ f e'm readngs are corrected by subtracting an enzyme blank comoosed o~f substrate and DNS reagent to which the diluted enzyme is added with immediate color development/quenching rather than incubation.

Comroonent Weiaht Percent ulu~zvme B IUGlycerol *Tergitol XD- Table 1 below shows the eclntstability of this formulation. The enzymta activity increase is within exoerimental error.

tI Tabl I Enzve: Srabiliztio-, in, Exm- Enzyme Activity (UU per ML)* Oricrinal Samole Room Temperature 80C 50 0

C

t 9170 9130 92 00 Thirty days at: the condition indicated.

Examole 2- Z xampl1e 1 was reoated usingi Du-lzvme F-B, however, Teraitol XD was substituted by Pluronice P-105,c which is a commercial non-ionic block- copolymer available from BASF Wyandotte Corooration The cl oud Point of this conDoly-,mer is 91 0 C solution in water) and 94 0 C CIO- solution in water). The average molecular weight of the surfactant is about 01,50o.

Table 2 shows, within exomerimentai error, the red~uction i n *stability of this formulation when compared to Example 1 which appears to be a function of PluronicO 1--10 com-nared to Tergitol XJ_ Stability is nonetheless better than enzymes without undr teseconitinswithout the stabilization providdb Table 2 Wd Enzymue Stabilization In Examole 2 Enzym~e Activity (IU ,o-r ML)* Oricainal Samule Room TeMD erature 8 0 C 50 0

C

8400 8280 8970 7370 Thirty days at the condition indicated.

Examole 3- Examplec 1 was repeated using a protease enzyme from Solvay Enzymes, Inc. or a linase en'zyme from Novo Nordisk Bioindustrials, Inc., the results of Which are set forth in Table 3.

20

U

3:

I

I

Table 3 HT-Proteolvt.c L-1-750 (nrotease) Glycerol nlius) Tergitol IXD Akctivity (14 days) at 50 0

C

at Ro om Temn (2 0 0

C)

weia'-t Comoonent Resinase A2X' (lipase) Glycerol (96k plus) Tergitol XD Water PluronicO P105 Tetronic -13040 BASF Wvandotte

SOOC

at Room Temo. (200C) it will be aonarent to 91 .Wei c- t 0 .04A-9 0.033 0.048 0.067 those skill-ea in 0.0471 0.054 the art 0.0553 0. 0472 that modif:ications and variations can be made in the method and comoosition of the Dresent invention without departing from the spnirit or scone thereof". it is intended that these modifi;cations and Variations and their equivalents are to be included as nart of this invention urovided they come within the scone of the appended claims.

N

Claims (27)

1. 2. The method of claim. 1. wherein said enzyme is stabilized aainst decomposition at elevated temperatures bv said non-ionic polyether-npolvol bloc'k-couolymer surfactant which has a cloud point greater- than said temoeratures.
2. 3. The method of claim wherein said temneratures-are from about OOC to about 100 0 C.
3. 4. The mnethod of claim 1, wherein said enzyme is a system or an enz-.LL'e in comaoanaclon with water saidenzVTP Deifl stLai- lized against decoimposizion ffrom water by said non-ionic polyether-polyol block-cooolym-errsurr4 actant which raises viscositv of water in said system. The method or claim I, wherein said water is nresant in an amount from about: 1 to about 90 oreent by weighnt based on said enzymte and said water. The method of claim 2, wherein said non-ionic Unolyether-polyol block-copolymer surfactant is dissolved in an .orgranic solvent comnatible with Said enzyme.
4. 7. The method or c-laam wherein said non-ionic polyether-polyol block-copolymer surfactant is dissolved'in an organic solvent compatible with said enzyme.
5. 8. The method ofE claim 6 or 7, wherein- said solvent i hvdroohili1c.
6. 9. The method of claim 8, wherein said solvent is'a polyol or a mixture of nolvols. The method of claim 9, wherein said polvol has from 2 to about 6 carbon atoms and from 2 to about six hydroxyl groups. I- The method of claim wherein said enzymes are pro-: tease, amylase, cellulase, hemicellulase. ectinase, -22- 0-giucanase, lipase, ohospholipase, clucose isomte-ase, aThcose oxidase arid linoxidase-
7. 12- T-he method. oz apy one of claims 2, 3 or A- whri said non-ionic ipolyether-polyol block -copolymer surfactant is a uolyoxyalkyliene glycol ether all-block, block- hterc, heteric- block or heteric-'r'ttric block copolymer where said alkvlene units have fro-m 2 t.o about carbon asuil.
8. 13. The metnod of claim. 12, wherein. said nolvox-syalkyleie glycol ether contains hvaroohobnic and hvdroohilic blocks based on at leastL oxvethylene groups, oxyprzopylene groups or mix-Lures of said groups.
9. 14. The method of- claim 13, wheein the average molecular- I ;**weight of said polyoxyalkylene glycol ether is from about 500 to abDout 30,000 the weight ratio or the hvdroonoooe to the hydrophile of said polyoxyalkyle-ne gl-vcol ether is from about 0-4:1 to about 2-5:1 and the cloud uor~nt of' said Dolyoxyilkyleie glvcol ether ic from about OOC to about 100 0 C. An enzyme comoosition or matter stabilized against loss of activity at e-vtdtmeatures or water where said composition is an enzyme in combination with a stabilizing amount of a non-ionic polveather-o-olyvol block-copolymer surfactant 1.The composit-o of caL1, rein said enzyme is stabilized ag-ainst decomosition at elevated temneratures b)y t i:~:said non-ionic nolyethIer-nolyol block-copolymer surfactant which has a cloud point greater than said temperatures.
10. 17- The composition of claim 16, wherein said temneratures are from about 0 0 C to about 100 0 C.
11. 18. The compiosition of claim 15, wherein said enzyme is a sseiof anenzyme in combfination with water, said enzyme bein stabilized against decomposition from water- by said non-ionic polyether-polvol block-copolymer surfactant which raises te viscosity ofc water in sa id systeM. 1.Tecom osizion of- clam, 18, wh neren s ai Wa t er Drese-nt i an amou-nt fram, abour I to abour 9 0 necn by weicghr based on said enzyme and said water. The coMtDosition of claim. 16, wherein said nonf-ioni2c uolvethe--Dolvol bloc"--couolvmer sur-factant i s dissolved in, an orcxanic solvent comnazible with said enzyme.
12. 21. The COMOOSIcion of claim 1-S, wherein said non-ionic DolyetLher-1-olvol bloc k-conolymer surfactant is dissol-ved in an organic solvent compatible with said enzyme.
13. 22. The composition of claim 20 or 21, wherein said solvent: is hvaxropnrlic-
14. 23. The coTIDosrE2.on of claim 22, wherein said solvent: is a Dolvol or mixture or nolvolS
15. 24. The Ioj~sco orcam 23, wnerein said novol ha tro-m 2 to about 6 carbon atoms and from 2 to about 6 hydroxy2. groups.
16. 25. The composition or claim 15, wherein said enzymes are prot-ease, amylase, cellulase, hemicellulase, oectinase, -glucanase, lipase, ohosrholipase, glucose isomerase, glucose oxidase and linoxiaase.
17. 26. The comoosizion' or any one- of claims 16, 17or 18, *wherein saida on-ionlc nolvether-oolvol block-couolvmer surfactan't is a polvoxv-alkvlene glycol ether all-block, block- heteric, heteric-block or heteric-heteric block-conolyr-ner where said alkylene units have from 2 to about 4 carbon atoms.
18. 27. The composition of claim 26, wherein said uolyoxyalkylene glycol ether contains hydrophobic and hydrophilic blocks, each block being based on at least oxvethylene groups, or oxyoropylene groups or mixtures or said groups-
19. 28. The composition or claim 27, wherein the average molecular weight of said polyoxyalkylene glycol is from about 500 to about 30,000, the weight ratio of hydrophobe to hydrouhile of said polyoxvy glycol ether is from about 0-4:1 to ~7F about 2:1and hecloud point of said pDolV3xCVn1kVyFln a'ehe iS zrorn about- 0 0 C to about 100 0 C.
20. 29- The comoositaon of claim 20 where said molvether-nolvol bilock-comolvnie -is a polyoxy.alkylene glycol ether block- cboolvmazr having a hnydropnoobe based on a hydrocarbon morietv of an. alioh*Iatic 1monlohvdr-ic alcohol containing f-rom 1- to about 6 carbon atoms,_ where the nvdrocaroon moiety has attached thereto throucun an etn-er oxy an linkage, a heteric mixed chain ooxvethvlene and.i,2oxvoroDviene groups, the weight ratio or oxyethylene groups -to 1. 2-oxvnro~ov~ene groups in the hydroprobe rs _trort about 5::95 to about !1:5:85 and-the average molecular weight of the hvdroohobe IS from about 1,000 to about 2,000, a -vd-ona- bah attached to th OC n sbased on oxyethylene c= u th~ weight ratio ofhdohile to hydronhobe is from about 0.8:1 to about 1.2:1. The commosition o= claim 29 optionally including as a solvent, a uolvol having from. 2 to about 6 carbon atoms and from 2 to about 6 hvdr-oxvl groups.
21. 31. The camuctsitiozn of claim 30 wherein said enzymes are protease, amylase, ceIlulase, hemicellulase, nectinase, fl-glucanase, lipase, Dhosoholipase, glucose isomerase, glucose .*oxidase aand linoxidase-
22. 32. The composition or- claim 3i wnereirn saic solvent is glyzerol and said enzy-mes are amyvlase, protease or linase.
23. 33- The comnosition of claim 20 where said polyether-polyol block-conolymer is a nolyoxyalkylene glycol ether block- copolymer having a hydrouhobe based on a propylene oxide adducc of' propylene glycol where tne propylene glycol has attached thereto through an ether oxygen linkage, oxyoronviene groups, a hydrophile being attached to the hydropoe and is based on oxvethviene groups, the average molecular weight o f the surfactant is from !,100 to about 12,600, and the HLB is from about I-7 to greater than about 24- A9
24. 34. The comoosit-ion or claimn 33 ontionaliv including as a solvent, a nolyol havinq from 2 Lo about 6 carbon atoms and from 2 to about 6 hydroxyl grouns. The composition of claim 34 wherein said enzymes are protease, amylase, cellulase, hemicellulase, Dectinase, f-glucanase, lipase, uhospholipase, glucose isomerase, aliucose oxidase and liooxidase.
25. 36. The composition of claim 35 wherein said solvent is glycerol and said enzym~es are amylase, protease or lipase.
26. 37. The comos-ition of claim 20 where said polyether-po lyol block-conolymer is a polyoxyalkylene glycol ether block-copolvmer having a hydronhobe based on a propylene S**oxide adduct of ethylrene diamine where the ethylenediamine has attached thereto through an ether oxygen linkage, l,2oXypropy.Lene groups, a hydro-phile being attached to the mixed chain and is based on oxyethylene groups, the average molecular weight of the surfactant is from about 1,650 to about 30,000, and the 1ILB is from about 1-7 to greater than about 24.
27. 38. The composition of claim 37 optionally containing as a solvent, a polyol having from 2 to about 6 carbon atoms and from 2to about 6 hydroxcyl groups. The comnosition of claim 38 wherein said enzymes are protease, amylase, cellulase, hemicellulase, pectinase, f-glucanase, lipase, phospholipase, glucose isomerase, glucose oxidase and lipoxidase. The composition of claim 39 wherein said solvent is g lycerol and said enzymes are amylase, protease or lipase. dated thiS 15th day of February, 1999 BUCIMANq LABORATORIES INE-RNATIONXAL, INC. rwAT-R-iARK PATENT TRADEIARE( ATTORNEYS 2nd Floor, 290 Rurwocxi Road, HM~rHORN. VICIORIA 3122-