Background of the invention This application is a continuation-in-part of my co-pending application U.S.S.N. 508,476, filed 27 June 1983.

This invention relates to polymeric derivatives of naturally occurring chitin and to methods for making and using those derivatives.

Chitin refers to carbohydrate polymers of N-acetylated glucosamine monomer units synthesized in nature in plentiful supply, e.g., as part of the skeleton or shell of crustaceans and other arthropods, of mollusks, or in the walls of fungal cells. Naturally occurring chitin performs an exceedingly wide variety of architectural functions.

For some time it has been known that chitin's naturally occurring structure provides an unusual combination of properties including high tensile strength, toughness, bioactivity, biodegradability, and non-i munogenicity. A wide range of industrial uses has been suggested for chitin or its deacylated derivative, chitosan. [See, e.g., Muzzarelli, Chitin (1977) Pergamon Press, New York, N.Y. at pp. 207-265; Austin et al. (1981) Science 212; 749-753; and Rawls (1984) Chemical & Engineering News, May 14, pp. 42-45] While the estimated yearly biomass production of chitin (10 tons) is comparable to that of cellulose, its industrial promise generally has not been realized until now. This is so primarily because chitin from the most readily available sources, e.g., crustacean carapaces, occurs in intimate association including covalent bonding with other materials, such as protein, minerals (chiefly in form of calcium salts) , lipids, and coloring

matter (including carotenes) . All such extraneous matter must be removed prior to final preparation of chitin. Additionally, recovery and purification of useful chitin products has been hindered by the difficulty of dispersing or dissolving chitin in all but strong mineral acids_or exotic anhydrous solvents.

Thus, two difficult problems to be overcome to improve or enhance the commercial utility of chitin are separating the non-chitinous material from the chitin and dispersing the purified chitin in an aqueous solution so as to obtain the macromolecules in physico-chemically sufficiently uniform array as to make them more readily accessible for chemical and/or enzymatic manipulation. In sum, realization of chitin*s commercial promise requires a way to prepare a uniformly dispersed chitin product having strictly reproducible and predictable characteristics.

While considerable attention has been paid to chemical modification of various chitin-derived materials, the quality and preparation of the starting material used for such modifications has often been entirely ignored. For example, the starting material is typically referred to as "commercially available carbonate-free chitin" [Vogler U.S. Patent 2,831,851], "purified chitin" [Jones U.S. Patent 2,689,244], or "crude chitins... commercially available" [Stacey & Webber, Methods in Carbohydrate Chemistry, Vol. I, p. 228] .

Where a disclosure of the method used to obtain purified chitin is provided, it may be characterized as a "well-known procedure". Muzzarelli (pp. 89 et. seq.) provides a comprehensive review of various techniques that have been used for preparing chitin from raw material. BeMiller (1965) Methods Carbohydrate Chem. V.

"Chitin", pp 103-105 also reviews chitin preparations. Such preparations typically include crushing or milling to disperse the raw material in an aqueous solution, deproteinizing with hot concentrated alkali, and decalcifying with hydrochloric acid. In some cases the resulting material is then subjected to harsh oxidation during purification, for example by KMn0 ₄ , H ₂ 0 ₂ , or ozone. See, for example, Berger e_t al. (1958) , Biophys Biochem. Acta, 29Ϊ 522-534. Even where an attempt has been made to preserve some characteristic or other of naturally occurring chitin, the prior art ultimately has been forced to resort to relatively harsh conditions in order to disperse the chitin, as repeatedly acknowledged in the quotations given below.

One researcher approached the problem by trying to dissolve "commercial chitin" (of unknown origin) in acid systems such as trichloracetic/formiσ acid [Austin U.S. Patent 3,892,731] or in a combination of a chloroalcohol [e.g. chloroethanol] and a mineral acid (e.g. HC1, HNO-, H ₂ S0 ₄ , or H ₃ P0 ₄ ) [Austin U.S. Patent 3,879,377].

In later work, the same researcher turned to other solvents such as dimethylacetamide, N-methylpyrrolidone or mixtures thereof with an admixture of lithium chloride, because the procedures disclosed in the above-mentioned *731 and '377 patents provide "solutions which are not as stable as desired for storage for considerable lengths of time." [Austin U.S. Patent 4,059,457].

In yet another approach [Austin e_t al. U.S. Patent 4,029,727] the goal is stated to be "a solution of chitin in which the chitin is fully dissolved or dispersed, and without order or organization." To that

end, red crab chitin is dissolved in solvent systems comprising trichloroacetic acid, chloralhydrate, and methylene chloride.

Finally, Austin et al. U.S. Patent 4,286,087 proposes a process for treating chitin to make it easier to store and ship, while preserving the levo(-)optical rotation said to be beneficial in uses such as wound healing and supposedly characteristic of naturally occurring chitin. The process includes boiling chitin (e.g. from brown shrimp) for 1-1/2 hours in a mixture of 85% H-PO. and 2-propanol. Austin et al. (1981) Science 212; 749-752 discloses a similar procedure for making a redispersible chitin powder in which a chitin slurry is boiled for two hours in 85% H ₃ P0 ₄ and 2-propanol, dispersed in water, sheared, and freeze-dried. The phosphoric acid is said to be "held in the form of a phosphate salt by several free amine groups, since exhaustive water extraction and attempted elution with aqueous hydrochloric acid reduced the phosphorus content to 0.1 percent."

Another research group has taken various approaches to the problem of recovery of useful material from naturally occurring chitin/mineral/protein matrices. Peniston U.S. Patent 3,533,940 discloses preparing chitosan from crab shell by treating the shell with HC1, deproteinizing and treating it with permanganate and oxalic acid, following which the chitin is partially deacetylated with 40% caustic soda at 150°C. Peniston e_t al. U.S. Patent 4,066,735, discloses decalcification using excess sulfurous acid, said to be preferable to HC1 since the latter is relatively expensive and hazardous, and may be so strong as to "cause some degradation of chitin during demineralization treatment." Sulfurous acid is also

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well-characterized or consistent, and the digestions may not go to completion. Microscopic appearance of chitin isolated following established procedures is irregularly compacted (c.f. Fig. 3) and is thus at variance with the well-ordered appearance of chitin strands _iι situ (c.f. Neville infra and Richards, The Integument of Arthropods, Univ. Minnesota Press, 1951) . In addition, chitin materials prepared according to such techniques may be relatively compact and dense, as reflected in their ability to produce relatively high-concentration suspensions in solvent.systems, e.g. the 10% w/v solution used for determining optical rotation.

The extensive, long-standing, and continuing efforts to develop a procedure for preparing commercially useful chitin or derivatives such as chitosan with reproducibly consistent properties attest to the difficulty of the problems encountered. Moreover, existing methods for recovering deproteinized, decalcified chitin yield materials which are often unusable for industrial/agricultural applications. For example, the properties of chitosans derived from such chitin preparations also differ in their behavior with respect e.g. to sequestering of heavy metals which, though generally held to be a typical property of chitosan, nevertheless quantitatively varies widely vis-a-vis a given ionic species when products of successive production runs are tested (Proceed. II Internat. Confer. Chitin/Chitosan, Sapporo, Japan, 1982) . Thus, properties of such preparations are neither consistent nor uniform.

It has generally been assumed that such problems are inherent in the structure of the chitin and/or that they arise of necessity in the purification process and, therefore, that the products of

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demineralization and/or deproteinization are necessarily variable, inconsistent, and generally unreactive.

It is further assumed that "Chitin ... is not attacked by bacteria and it is difficultly hydrolized [Muzzarelli U.S. Patent 3,635,818];" and that chitin is "extremely resistant to enzymatic action [Dunn U.S. Patent 3,847,897] ."

"Chitin does not occur in its pure form in- nature but is usually associated with substantial quantities of protein and inorganic salts such as calcium carbonate. ... It should be noted that chitin is normally found in and associated with living organisms and would be expected to exhibit altered- characteristics when removed from the ambit of the biological processes [Silver U. S. Patent 4,120,933].

Similarly, another researcher has said,

"Because of its insolubility and its close association with other substances, [chitin's] isolation requires the use of drastic methods to remove contaminating substances; these methods probably degrade chitin to some extent." BeMiller (supra) .

Attempts to circumvent these problems by utilizing crustacean meal made from cannery waste have also not met with success. According to Peniston [U.S. Patent 4,199,496] :

"In general, shellfish waste meals have limited markets due to their high mineral and chitin content. This limits levels at which...shellfish waste meal can be fed to farm animals and to poultry." and "Chitin is indigestible for poultry and livestock and can cause intestinal irritation. Thus, only the protein [of crustacean waste meal] is of real value as a feed material and the other components of shellfish- meal are undesirable diluents detracting from the feed value."

Summary of the Invention I have discovered that naturally occurring molecular chitin structures are complex and highly ordered, as well as extremely fragile, and sensitive to many chemical treatments and that existing efforts to remove' mineral, protein, and other matter associated with naturally occurring calcium-containing chitins radically alter the native structure. Specifically, native calcium-containing chitins are present in a matrix, parts of which include chitin-protein and protein-protein cross-links. The matrix is heavily encrusted with minerals such as calcium salts, and often includes lipids and coloring material. Enzymes which are specific to chitins are extremely sensitive to the fine structure of the chitins. Once lost, that fine structure cannot be regenerated from chitin that has been altered during the process of separation from its natural environment.

The invention features fibrous, dispersible chitin polymers which have been purified in that they have been substantially dissociated from the native chitins* calcium-reinforced protein matrix. The polymers are stabilized by sparse esterification, in

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that they retain the structural features of the native chitin as evidenced by rapid and quantitative recognition by enzymes specific for naturally occurring chitins including chitinase (EC 3.2.1.14) and chitin deacetylase, and complete lack of recognition by enzymes specific for other substrates, e.g. lysozymes.

By sparsely esterified I mean that the ratio of cross-linking ester functions to sugar residues is between 1/500 and 1/1500 most preferably about 1/1000. By fibrous, I mean that the isolated or dispersed chitin is predominantly characterized by relatively large, well organized elongated fibers, for example, thick fibers that are between about 1 and 3 microns in diameter. By dispersible, I mean that the chitin polymers can be readily and uniformly suspended in an aqueous solution. By chitin-speci iσ enzymes, I mean enzymes such as endo- or exo-chitinases or chitin deacetylases which react rapidly and quantitatively with naturally occurring chitins, and mean to exclude enzymes, such as non-specific hexosaminidases or lysozymes, which recognize structurally similar but not necessarily identical glycosidic bonds, and which react slowly, if at all, with chitin that largely retains its natural molecular structure. in preferred embodiments of the dispersed chitin polymer, the ester functions are sulfate or phosphate functions assumed to form cross-links between chains of N-acetylglucosamine units; or the ester functions are aσetyl or nitrate functions which, though not cross-linking, nevertheless stabilize the naturally occurring fibers; and the thick elongated fibers are between 1-3 microns in diameter.

The invention also features a method of preparing the above-described chitin polymer from a

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naturally occurring calcium-reinforced chitin protein matrix in which the matrix is first decalcified and deproteinized, and then the chitin is dispersed and stabilized in an ester-forming acid before being recovered.

In preferred embodiments of the method, the temperature of the chitin dispersal medium is kept uniform to within +1° C (most preferably 4^0.5° C) throughout the dispersal; the temperature of the dispersal is kept below 8°C; the dispersal is continued for between 3-48 hours; and the acid is a multi-ester forming acid (such as sulfuric or phosphoric acid) which is pre-chilled and added slowly to the aqueous raw-chitin suspension to a final concentration of less than 90%. ipids and other non-chitin material (such as coloring material) are removed in the course of the preparation, without steps introduced specifically for this purpose, since such steps frequently are damaging to the- delicate chitin fine structure. Because the resulting chitin polymer products substantially preserve the native chitins' structural features, they are particularly useful as a substrate for enzymatic processes, and will perform well in selected chemical procedures. In this, they contrast sharply with existing purified and/or regenerated chitins which exhibit collapse of innate structure by greatly altered densities, greatly altered appearance in light and/or electron microscopic examination, and inability to form uniform suspensions in aqueous solutions and/or to furnish derivatives with predictable properties and/or to serve as substrates for specific enzymatic processes.

The invention thus features the use of the chitin polymers in a number of enzymatic processes:

formation of structured chitosan by enzymatic deacetylation of stabilized chitin; fertilization of plants by enzymatic degradation of the stabilized dispersed chitin polymer to provide a steady, predictable supply of useful (i.e. "fixed") nitrogen without itself being subject to loss by leaching; use as a source of non-protein nitrogen (NPN) and sugar and acetate carbon, i.e. energy as well as NPN, in ruminant, bird., or fish feedstuffs; formation of a nutrient lawn containing the chitin polymer which is used as an indicator to detect chitinase, e.g. to select microorganisms that produce extracellular chitinase; and as a source of ammonia to be trapped and exploited for fertilizer formulations in industrial fermentations, while the residual fructose can readily be industrially fermented to ethanol—a desirable source of energy, e.g. for operating internal combustion engines.

The importance of a process that reproducibly yields a predictable product is illustrated by the use of chitin as a ruminant feed supplement. Ruminants utilize nitrogenous compounds by microbial fermentation and biosynthesis, e.g. of amino acids, in the rumen. Chitin that is predictably recognized by specific microbial enzymes can furnish non-protein nitrogen for augmenting such relatively incomplete feeds such as hay, especially in winter when a diet of hay alone leads to a negative nitrogen balance in the animal. However, if such enzymatic recognition cannot be assured, chitin cannot be used as a feed, because undigested chitin remaining for more than a few days in the stomach is extremely detrimental to the animal. Thus, processes at present practiced which yield products of uncertain structure and composition are of no commercial value.

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Apart from the enzymatic uses specified above, the chitin polymer or chitosan produced from it are generally useful as industrial feedstocks where consistent, predictable properties of native chitin are important. Specific examples of uses proposed for chitin and chitosan include: agents to selectively chelate various metal ions (e.g., for removal of radioactive ions that are products of nuclear fission, to purify metal-working waste water, to reclaim metal waste, or to purify reaction products) ; paper or fabric additives or finishes which carry dye, repel soil, shrink-proof, etc.; a photographic substrate; adhesives; a leather finishing agent; a formative material for medical membranes; a pharmaceutical carrier for poorly soluble drugs; a blood coagulant; a biodegradable medical filament or fabric; or an adjuvant for arable land to improve tilth while at the same time providing significant usable nitrogen for plants.

The following references disclose methods of using the chitin polymer and chitosan derived from it: Jones U.S. Patent 2,689,244—thickener for paste, adhesive, additive for drilling; Austin U.S. Patent 4,286,087—wound healing; Austin 3,879,377—pape -making, surfactant; Vogler U.S. Patent 2,831,851—blood anticoagulant; Dunn U.S. Patent

3,847,897—thickener, stabilizer in food; Cushing U.S. Patent 2,755,275 blood anticoagulant; Delangre U.S. Patent 2,842,049—photo processing; Peniston U.S. Patent 3,533,940—anti-coagulant; Muzzarelli U.S. Patent 3,635,818—metal ion chelating; Bridgeford U.S. Patent 3,689,466—soil repellant; Balassa U.S. Patent 3,914,4130— ound healing; Katz U.S; atent 3,940,317—isolating lysozyme^ Dunn U.S. Patent 4,034,121—food thickener; Slagel U.S. Patent

4,056,432—paper additive; Nieuwenhuis U.S. Patent 4,156,647--wastewater treatment; Casey U.S. Patent 4,068,757—powder for surgical gloves; Capozza U.S. Patent 4,074,713—surgical elements; Muralidhara U.S. Patent 4,293,098—animal feed additive; Silver U.S.

Patent 4,120,933—removal of radioactive waste; Schanze U.S. Patent 4,357,358--animal feed additive. The above patents are hereby incorporated by reference.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiment and from the claims.

Description of the preferred Embodiment I will next briefly describe the drawings of the preferred embodiment. Drawings

Fig. 1 is a photograph (400x) of stabilized shrimp chitin. .

Fig. 2 is a photograph (400x) of stabilized lobster chitin.

Fig. 3 is a photograph (400x) of de-stabilized

(collapsed) crab chitin

Fig. 4 is a photograph (9x) of a chitin-agar lawn exposed to extracellular chitinase exuded by the cells shown during, and as a result of, their growth as colonies.

I. The Native Chitin.

Virtually any recognized natural source of calcium-bearing chitin serves as an appropriate starting material for production of purified stabilized chitin. Crustacean shells and whole crustaceans (e.g. krill) are among the most plentiful sources of such native chitin.

II. Preparation of Stabilized Chitin.

A. De-Calcification and De-Proteinization Intact chitin-bea ing structures are initially preserved by boiling in water or rinsing with an antioxidant solution, and then air dried. This allows storage at any convenient ambient temperature of the material, e.g., cannery waste shells, or transport to processing plants, without spoilage, and at greatly reduced weight. The structures are then mechanically disintegrated by cutting, milling, or grinding to a uniform size for further processing; this step may equally well be performed after decalcification, as discussed below. Calcium (present largely as CaC0_) is removed by treatment with dilute hydrochloric acid, EDTA, or enzymatic action. If HC1 is used, decalcification must be done before deproteinization (described below) because any exposure of the deproteinized prestabilized chitin to HC1 or other non-esterifying acids such as HCIO, will irreversibly destroy its native structure.

Following decalcification, proteinaceous material, lipids, and coloring material may then be removed using boiling dilute NaOH or specific enzymes. Once these materials are removed, the innate chitin structure is vulnerable to collapse, particularly if exposed to non-este ifying acids and/or oxidizing chemicals. Such collapse is irreversible and extremely detrimental, in that collapse renders the material unsuitable for hydrolysis by specific chitin-recognizing enzymes; it dramatically reduces the_ consistency and quality of performance of the isolated material in most applications. ' ^{" ■}

In sum, it is desirable to maintain the deproteinized chitin in a relatively neutral environment (e.g. between pH 5 and 8, or if desired in the dry state at any convenient ambient temperature) until the next step (the dispersal step) is performed, it is particularly important at this stage to avoid exposure to non-esterifying acids (e.g. HCl) and/or to strongly oxidizing conditions (e.g. permanganate solution) .

B. Stabilization and Dispersal of ' Purified. Chitin

Extreme care must be exercised to disperse the resulting decalcified and deproteinized chitin to avoid damage to its structure as described above. Specifically, the temperature of the dispersion, the final concentration and rate of addition of the acid, and, most importantly, the anion of the acid used, must be controlled with precision. Acid dispersal is required so as to create a substrate that is rapidly attacked by chitin-specific enzymes; mere milling is insufficient to reduce the macromolecular assemblies to a size which enzymes can readily attack.

While not being bound to a particular theory, it appears that the acid used must be one capable of esterifying the chitin. Most preferably, the acid should be multi-ester forming (i. e., its corresponding base should have more than one oxygen available for ester formation) . Examples of such acids are sulfuriσ acid and phosphoric acid. The sulfate anion has two free oxygens which are sufficiently nucleophilic to form ester bonds. If the sulfur-bonded oxygens bond to separate chitin fibrils, they can form a cross-link between them, and, if sufficient but not too many cross-links are formed, the chitin fiber structure will be stabilized. Such stabilization remains effective so

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long as the stabilized chitin is not subjected to conditions that hydrolyze the ester bonds. If those bonds are hydrolyzed (e.g. by subjecting the chitin to prolonged exposure to acidic conditions, above 25% (V/V) or to high heat together with highly basic conditions as customarily practiced in chemical deacetylation of chitin during the manufacture of chitosan) , the native chitin structure collapses irreversibly.

During dispersal, if the acid concentration is too great relative to the water concentration, an apparent over-este ification ^" occurs resulting in a product of no discernible practical value. If the acid concentration is- too low, and/or rate of acid infusion occurs at too slow a rate, the bonding (cross-linking) may not keep pace with dispersal, leading to collapse of native structure, or dispersal may fail to occur. Excessive temperature during dispersal, even if only in limited locations, or prolonged acid contact again destroy the native chitin structures as well as reducing yield, presumably through excessive chain shortening. Finally, exposure of the material to strong oxidizing ^• agents such as ozone, H ₂ 0 ₂ , or permanganate (e.g. as often done to remove color-forming material) must be avoided to prevent degradation of chain length as well as destruction of innate structure and sugar monomers. In sum, during dispersal, the temperature of the dispersal medium should be controlled throughout the medium to within at least 2°C and most preferably 1°C, and the dispersal medium should be restricted to aqueous ester-forming acids in suitable intermediate and final concentration. To avoid localized "burns" from excessive temperature or acid concentration, the acid should be pre-cooled and added slowly with stirring to the precooled stirring suspension of crushed or milled chitin in the corresponding amount of water.

The preferred temperature of the pre-cooled acid, and throughout the dispersal medium, is below 8°C and most preferably below 4°C; the temperature should be greater than -5°C to prevent freezing. Initially, the acid concentration is below about 80% (V/V) , and most preferably is below 20%. Acid is added up to a final concentration of between 25 and 90% (V/V) and is most preferably less than 70% (V/V) because yield is significantly reduced above that level. The time of exposure of the chitin to the dispersal acid is between

3 and 48 hours, and most preferably less than 24 hours.

After dispersal and esterification, the stabilized product is precipitated in cold rapidly-stirred water, alcohol, or a similar organic solvent which optionally is admixed with water, and harvested. _. Acid is removed by repeated water -rinses, and the product is preserved by adding azide, unless it is to be used in manufacture of comestibles.

Figs. 1 and 2 are 400X photographs of stabilized sparsely esterified shrimp and lobster chitin, respectively, prepared according to the described method.

C. Example Of Laboratory Chitin Preparation

Shrimp chitin, when decalcified by cool dilute HCl and deproteinized by repeated 30 minute boiling in

IN NaOH, is crumbly and easily disintegrates. In a typical experiment, 4 gm of such material is suspended in 100 ml water at 4°C while 100 ml cone. H„SO _Λ at

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4°C is added to the stirring suspension at the rate of 1 drop/30sec. When all chitin has dispersed, the viscous solution is precipitated in rapidly stirring 50% aqueous ethanol at 4°C, harvested in the centrifuge, and washed to pH 5. After taring, a suspension of 6 mg/ml is prepared in 0.1 M phosphate-acetate buffer (pH 6.5) ; the

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buffered substrate is preserved with 0.02% azide or other similar preservative or mold-inhibiting substance, or, before or after buffering, the substrate may be freeze-dried for storage. The specific methods outlined in the above procedure are not intended to be restrictive with respect to modifications that may be dictated by upscaling to industrial production; for example it may be desirable to carry out the mixing procedure during the dispersal by sparging. Precipitation by addition of precipitant to the -.dispersal medium (rather than vice versa) may give better results in larger-scale operation. The precipitated solid may be harvested by a "sluicing" procedure which might combine continuous washing and harvesting.

III. Product Characterization A. General

The stabilized chitin resulting from the above process is substantially free of non-chitinous material, yet it substantially preserves the structure of the native chitin. By "substantially free of non-chitinous material," I mean that the product has less than (by weight) 2% amino acid and negligible ash content and is more than 98% chitinous material. This contrasts with chitins prepared by excessively harsh procedures, such as use of elevated temperatures or strong acids which destroy the naturally occurring fine structure, trapping amino acids and/or ash within the resulting dense mass of material. By the phrase "substantially preserves the structure of the native chitin" I mean that the product in question appears to retain fibrous structure that is visible in the lightf microscope and may preexist in similar form in the deproteinized carapace; I also mean

that it is recognized by enzymes that are specific for native chitin, such as insect molting fluid chitinase, Streptomyces chitinases and other specific hydrolytic enzymes, e.g. from wheat germ, or S_. arcescens; such enzymes react in variable fashion, or not at all, with substrates such as .chemically collapsed or otherwise structurally damaged chitin. Stabilized chitin is also unreactive with enzymes specific for other materials of known structure; for example, it is not susceptible to attack by lysozyme. More specifically, the stabilized chitin exhibits normal kinetic behavior with chitin-specific enzymes: it reacts at rates that are comparable to rates of in vivo chitin reactions such as rapid enzymatic hydrolysis typical of old cuticle in arthropod molting or breakdown of shed carapaces, e.g. in aquatic environments. Further, the kinetics of the reaction can readily be characterized (e.g. as to reaction order and mechanism) by standard techniques. In contrast, structurally collapsed chitin or chitin derivatized so as to be made water-soluble will be degraded slowly and abnormally by non-specific hexosaminidases, or may even be attacked by lysozymes and/or commercial preparations of beta-glucosidase, but either not at all, or else very slowly, by chitin-specific enzymes. Rates of degradations such as those listed above are so slow, and the kinetics of such reactions are so abnormal, that they cannot be indicative of true i _j i vivo reactions.

Even labeled products sold or prepared in the laboratory as substrates for testing alleged chitinase activity reflect abnormal enzyme kinetics. See Molano et al. (1977) Anal. Biochem. 8 : 648-656.

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- 20 - The chitin-specific enzymes which recognize the stabilized chitin polymer to the exclusion of structurally altered chitin derivatives include all enzymes known to recognize chitin in its naturally 5- occurring form such as true chitinases (not 'non-specific hexosaminidases or lysozymes) and chitin deacylases. Those enzymes include chitinases (EC 3.2.1.14) , such as those obtained from Streptomyces cultures and available from Sigma Chemical Co., St. Louis, Mo., and other

10 commercial sources; also included are enzymes from

Manduca sexta, or digestive tracts of arthropod-eating vertebrates and invertebrates. Other chitin-specific enzymes include the chitin deacetylases such as those discussed below.

15 Microscopic examination of the product resulting from the method of chitin purification attained above reveals fibrous structures. ^' Specifically, a thick (diameter from about 1-3 microns) fiber is observed; without being bound to a particular

20 theory, the thick fibers are believed to arise chiefly by cross-linking of thin, pre-existing fibers occasionally visible in tightly coiled form by microscopic examination of stabilized chitin preparations. The cross-linking is achieved by

25 polybasic ester functions as previously described. The thin fibers may correspond to the fiber structure seen as integral parts of the native chitin structure in electron micrographs at very high magnification [see, Neville, Biology of the Arthropod Cuticle, e.g., pp

30 170-174 Springer-Verlag, New York 1975].

These fiber structures can be altered, e.g. mechanically, in predictable ways, and such alterations affect, again predictably, their digestion by enzymes. If proper care has not been taken during preparation.

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the collapse of such thin fibers releases short (water-soluble) segments of chitin thought to arise from artifactual intra-fibrillar chain scission. Insect molting fluid chitinase, having both endo- and exochitinase activity, digests a mixture of thick and thin fibers completely; commercially obtained Streptomyces chitinase from Sigma Chemical Co., which in some preparations retains little or no endochitinase activity, may cleanly digest only the thick fibers, while the (collapsed) thin fibers are not attacked at all. These facts are easily established by examination with a light microscope. If, however, collapsed chitin, e.g. collapsed thin fibers or chemically collapsed material as shown in Fig 3, is exposed to a commercially available preparation of "beta-glucosidase" (which is not supposed to have any chitinase activity at all) , the collapsed material is attacked at a fairly rapid rate, with the thick fibers remaining intact. Microscopic examination further reveals that chitin "purified" by existing methods is, in contrast, not fully recognized by chitin-specific enzymes in that substantial residues, even those which microscopically resemble fibers but which have been assembled from chitin that has suffered initial destruction of fine structure with subsequent partial "regeneration", remain undigested for substantial periods (days, if not indefinitely) .

The ester character of the product is demonstrated by radioactive tracer experiments, by the use of different ester-forming acids, and by direct analysis for covalent bonding of ester residues, i.e., release of such ester functions only following complete hydrolysis of the stabilized chitin.

The ^" product is particularly characterized in its relatively non-dense and uncompact nature, as

evidenced by its behavior in suspension and solution in solvent systems, in that relatively small amounts of product can be suspended or dissolved in comparison, e.g. to the dense collapsed preparation described by Austin (e.g. in U.S. Patent 4,286,087).

B. Specific Tests For Chitinase Activity I have developed or adapted a variety of tests for measuring or detecting the various aspects of chitinase activity encountered using native stabilized fibrous chitin, and these will now briefly be described.

1. "Clearing assay." In this test, one quantitates the light scattered by suspended chitin particles. The amount of light scattered can be accurately measured as the ratio of incident light at 350nm wavelength that is not received by the phototube in the spectrophotometer. This ratio is highly sensitive to particle size and, within experimentally determined limits, is linearly related to it. "Clearing" i.e. the reduction in the ratio of light scattered, serves (with proper controls) to guantitate the degree of reduction in particle size resulting from the activity of endochitinase, i.e. enzymatic activity scissioning chitin particles, presumably at random, into shorter lengths. By contrast, the clearing test gives far less useful results when only exochitinase activity is present, i.e. when the activity to be measured consists entirely in the serial removal of short lengths, most typically disaccharides, from either the reducing or the non-reducing ends of the polysaccharide. The reaction set-up for the clearing test is as follows:

In 2.0 ml final volume, the following quantities of reactants are assembled:

50mM phosphate acetate buffer (pH 6.5) ImM CaCl ₂ 2.4 mg chitin/milliliter

0.1 ml enzyme/milliliter (from a stock solution containing 4.0 mg solid per milliliter) .

The reaction is initiated by addition of enzyme and incubation at 37° with stirring is continued for as long as desired. To take a reading, the mixture is poured into a cuvette; it is then immediately returned to the test tube and the incubation continued.

2. Colorimetric test for N-acetyglucosamine. Such a test is set up with the same reactant concentrations as given for the clearing test. Three test tubes containing 1 ml of total reaction mixture each are prepared for each experimental point desired. To assure the proper zero value, each reaction mixture is pre-stirred at 37° for a minimum of 5 minutes. Enzyme (e.g. Streptomyces chitinase from Sigma Co.) is then added. A zero-time sample is obtained by spinning one of each triple set of tubes at 12,000xg in a microcentrifuge and sampling 0.4 ml of the supernatant. The remaining tubes from each set are incubated with continual stirring at 37°for 30 minutes, at which time 0.4 ml samples are taken in the same manner.

To each 0.4 ml sample, 0.1 ml excess hexosaminidase solution is added and the samples are incubated at 37° for a further 15 minutes to convert any oligomeric products to the monomer N-acetylglucosamine. Duplicate sub-samples of 0.1 ml each are then taken and the colored adduct is developed quantitatively by a somewhat modified version of a well-known procedure (Reissig et al. (1955) J. Biol. Chem. 217: 959-966).

3. Qualitative Detection of Chitinases. The following two tests may be used to detect the presence of chitin-specific enzymes using a stabilized native fibrous chitin. 5 a) Chitin-agar plaque test. A chitin-agar

"lawn" is prepared as follows: 7 mg of stabilized native fibrous chitin is suspended in 15 ml of a hot sterile solution containing 1.8 gm agar dissolved in 100 ml of water or buffer. The hot suspension is poured _Q into a sterile petri dish of suitable size. The layered suspension sets quickly at room temperature and may then be used to plate out microorganisms. Any such organism adapted for the formation of extracellular soluble chitinase will be able to form colonies on the 5 chitin-agar; as the colony grows, the soluble chitinase diffuses outward from it so as to furnish necessary nutrients for further growth of each colony. This leads to the formation of a clear "halo" or "plague" around the colony which is easily distinguished from the 0 uniformly cloudy chitin suspension forming the original lawn (see Fig. 4) . b) "Abklatsch" test. This is a more general test for carbohydratases and works particularly well for cell-free enzymes. A carbohydrate-agar lawn is again 5 prepared in a petri dish as in the previous procedure. The chief requirement for this test, however, is that the carbohydrate be either very large (as e.g. chitin or chitosan) or else non-reducing (e.g. sucrose or trehalose) . The enzyme to be tested is spotted on the 0 set agar lawn, the agar is covered with a piece of moistened filter paper, and the assembly is incubated for a few hours to overnight. The filter paper is removed and airdried. Any small, hence diffusible

OMPΓ

reducing sugar resulting from enzyme activity will have diffused, both into the agar (where it is of course lost) and into the filter paper where it can be detected by the alkaline silver method for reducing sugars (Trevelyan and Harrison, Biochem, Jour. 50_: 298-303).

4. Uses of radioactive label. I utilize labeled compounds to determine enzyme recognition of stabilized native fibrous chitin. If a radiolabeled chitin precursor, e.g. N-acetylglucosamine, is injected during the time of maximal rate of synthesis of new cuticle chitin, i.e. a few hours prior to ecdysis, labeled chitin may be obtained from various arthropod sources. If, for example, such a chitin bears label only in the N-acetyl group, suspected deacetylases may be tested for by comparing solubilized label to label that continues to remain with the insoluble residue even after many washings. In this manner, extracts from both Rhizopus pseudochinensis and Mucor rouxii were shown to remove 25-30% of label within 24 hours, if the sugar is labeled in the ring either by 14C or non-exchangeable

₃

H, appearance of acid soluble label readily identifies the presence of chitinase activity. Another manner of introducing label is to use radiolabeled sulfur or phosphorus in the anions of the esterifying acids; such label is used to show directly that acid-dispersed stabilized chitin contains covalently bound ester functions, since label will be released only following (complete) hydrolysis of the labeled material.

5. Deacetylation. There are several methods for verifying recognition of native chitin substrate by deacetylases which yield chitosan [See IV. A. below] . One of the gross behavior patterns by which chitosan is

distinguished from chitin is in a rather coarse colorimetric behavior; a more reliable test consists in dissolving any chitosan. that may have formed in 3% acetic acid, in which chitin is wholly insoluble. A third test involves the use of chitin labeled in the acetyl group. Chitinases and chitosanases are different enzymes. By all the latter tests, chitosan retaining its chitin-like molecular architecture was produced from stabilized native fibrous chitin by specific deacetylases.

6. Other Tests. Still other tests were used to characterize the purity of the native fibrous chitin; those tests included: conventional and/or radiolabel tests with respect to protein content, sulfate content, amino acid .content, ash present, and/or phosphate content of various preparations.

Example The following example demonstrates a method of establishing that a particular chitin product is rapidly and quantitatively recognized by a chitin-specific enzyme.

Reaction mixtures are prepared containing the following in a total volume of 1.0 ml: 2.5 mg chitin 50 mM phosphate-acetate buffer (pH 6.5)

1 mM CaCl ₂ .

Reaction is initiated by addition of 10 microliter Manduca molting fluid; this can most advantageously be obtained by tapping pharate pupae at the proper time, and quickly freezing it in the presence of phenylthiourea to prevent melanin formation. The reaction mixtures are stirred continuously in a 37°C waterbath. Activity is then measured as the absorbanσe

difference in Morgan-Elson color, developed in 0.1 ml aliquots sampled at 5 minutes and 15 minutes of incubation time. Average activity measured is about 1 milligram of N-acetylglucosamine produced per minute per milliliter of raw molting fluid. As an indication of the previous lack of suitable substrates for chitin-specific enzymes, 1 unit of chitinase activity is defined in the 1983 Sigma Chemical Co. catalog as the production of 1 milligram N-acetylglucosamine equivalent in 48 hours, with 2-3 units claimed per milligram of solid for the Streptomyces chitinase sold commercially by this company, ith stabilized chitin, this same enzyme preparation can be shown to possess about 19 units per mg solid, while 1 ml raw Manduca molting fluid in these terms might contain in excess of 250,000 units. I consider reaction with a chitin-specific enzyme under the above conditions rapid, quantitative, and specific, if N-acetylglucosamine color that develops with a 0.1 ml subsample gives an appreciable rate of increase as a result of enzyme action (see section III.A.2, above) within 10 to 30 minutes. By "appreciable," I mean a change in absorbance of light at 585 nm of a minimum of 0.100 absorbance units; by "specific" I mean product generation that is linear with respect to time. A clearing test would be required to establish that the enzyme reaction is quantitative; under the conditions described for this assay, an opaque suspension of stabilized chitin would clear completely in under 1 hour. The above-described clearing test (III.B.l) and colorimetric test (III.B.2) were performed using commercially available (Sigma) Streptomyces chitinase on the following three substrates:

1) stone crab chitin that had been alternately decalcified (1 N HCl, 1 hour at room temperature) and deproteinized (2 boilings in NaOH) , after which those procedures were repeated before acid dispersal. The resulting material, as shown in Fig. 3 (400x) , is substantially collapsed into a dense mass lacking well-defined fine structure;

2) chitin prepared according to the procedure in U.S. Patent 4,286,087 for "reorganizing" chitin into a "microcrystalline" form by-dissolving commercial crustacean chitin, grade B, from Calbiochem-Behring in 85% H-PO. and 2-propanol; and

3) native stabilized fibrous shrimp chitin prepared by the method of the invention described above. As shown in Fig. 1, the resulting product was a dispersal of stabilized chitin fibers.

The "collapsed" and "regenerated" chitin (Nos. 1 and 2 , above) did not react to completion and reacted far more slowly than the native stabilized shrimp chitin, particularly with batches of chitinase having predominantly exochitinase activity.

IV. Use.

Generally, the stabilized chitin polymer can be used in any of a large number of ways listed above. Specific uses are as follows.

A. Deacetylation.

Chitin has a substantial percentage of its sugar groups N-acetylated; many of the uses described

above are applicable to ^" chitosan, which is largely deacetylated. The ability of stabilized chitin to serve as a suitable substrate for chitin-specific enzymes enables enzymatic production of stabilized chitosan from stabilized chitin.

Specifically, deacetylases obtained from the following species can be used to enzymatically deacetylate stabilized chitin: Rhizopus pseudochinehsis; Rhizopus oligosporus; and Mucor rouxii. Specifically, cell extracts can be used. A reduction in acetylation by 25-30% from an initial acetylation of about 90% (e.g. from about 90% to 65%) will yield a preparation within the range of maximal chitosanase activity. Chitosans produced enzymatically from stabilized chitins will have predictable and reproducible properties; for example, a particular formulation will sequester specific heavy metal ions in predictable reproducible fashion.

B. Nitrogenous Soil Adjuvant and Fertilizer.

Chitinases degrade stabilized chitin to oligomers or monomers of N-acetylglucosamine; further degradation by microbial enzymes into e.g. ammonia has long been known. [See, for example, Davidson, E.A. , "Metabolism of Amino Sugars", The Amino Sugars (Balasz et al., Eds) Vol. IIB pp. 1-44 (New York: Acad. Press, 1966)]. It has also long been established that chitin, like other polysaccharides, tends to take up water because of the affinity of free hydroxyl groups for water molecules. Thus, incorporating stabilized chitin into soil can serve several purposes: it can improve tilth, and can also serve as substrate in the slow

release of "fixed" nitrogen in a form either directly assimilable by plants, or to be taken up by plants following microbial oxidation, e.g. to nitrate. Use of stabilized chitin for this purpqse can also be very advantageous because of its total insolubility in all aqueous solutions one might expect to find in soil. Still another advantage to crop plants would accrue from the fact that chitin tends to support growth of soil actinomycetes which help to keep down plant parasites, at least in part by exuding antibiotics.

It has been estimated that proven reserves of natural gas in the United States will be exhausted by 1990. Natural gas forms the industrial feedstock for the nitrogenous fertilizer industry; use of stabilized chitin could thus fill a gap that would otherwise have to be filled by expensive imports.

C. Ethanol Production.

^{' "} As described above, stabilized chitin can serve as feedstock in well-understood industrial microbial fermentations. Fixed nitrogen can be trapped as chitin is degraded, first to N-acetylglucosamine and then to fructose. The latter can then serve as feedstock for the production of ethanol, a clean and efficient energy source, e.g. for internal combustion engines. In short, the native stabilized fibrous chitin thus will serve as a vast source of energy from renewable biomass..

D. Feed Supplement.

As described above, the native stabilized fibrous chitin can be added to feed of ruminants (or other animals, such as commercially raised birds or fish) to serve as both a nitrogen source and an energy source by providing acetyl groups, fructose monomers.

and nitrogen in assimilable form. The stabilized chitin slurry (or dried components thereof) can be combined with conventional feed. The above-described enzymatic tests can be used to determine the nature and presence of enzymes which will consistently and rapidly digest the stabilized chitin. Knowledge of the stoichiometric composition of the chitin can be used to determine, together with the needs of the animals in question and the amount of other feed used, the amount of chitin to add to the feed.

Other embodiments are within the following claims.