WO2000024874A1

WO2000024874A1 - Recombinant extracellular chitinases and uses thereof

Info

Publication number: WO2000024874A1
Application number: PCT/US1999/025180
Authority: WO
Inventors: Alexey Fomenkov; Nemat O. Keyhani; Saul Roseman
Original assignee: Johns Hopkins University
Priority date: 1998-10-28
Filing date: 1999-10-27
Publication date: 2000-05-04
Also published as: EP1127103A4; WO2000024874A9; JP2002528072A; MXPA01004251A; CA2347373A1; AU1599500A; EP1127103A1

Abstract

The present invention features isolated polynucleotides and polypeptides that relate to extracellular chitinase (E-Chitinase) from a marine organism and particularly certain Vibrio and especially Vibrio furnissii. Also featured are methods of using the isolated polynucleotides and polypeptides to make specific chitin oligosacharrides.

Description

RECOMBINANT EXTRACELLULAR CHITINASES AND USES THEREOF

Funding for the present invention was provided in part by the Government of the United States by virtue of Grant No. GM51215 from the National Institutes of Health and Grant No. NA46RG091 from the National Oceanic and Atmospheric Administration to the Maryland Sea Grant. Accordingly, the Government of the United States has certain rights in and to the invention claimed herein.

1. Field of the Invention

The present invention generally relates to chitin catabolizing proteins and particularly to extracellular chitinases (E-chitinases) obtained from specific marine bacteria including recombinant E-chitinases and fragments and derivatives thereof.

Further included are polynucleotides encoding the E-chitinases as well as fragments and derivatives thereof. The present invention has a variety of applications including use in the preparation of chitin oligosaccharides.

2. Background of the Invention

Chitin is an organic polymer of substantial natural abundance. In particular, chitin is a homopolymer consisting of covalently linked β, 1→ N-acetylglucosamine

(GlcNac) residues. See e.g., Nalin, D.R. (1976) Lancet 2: 958; and Nalin, D.R. et al.,

(1979) Infection and Immunity 25: 768.

The utility of chitin and related compounds has been recognized in several fields including medicine, agriculture, research, and industry. For example, it has been reported that chitin is useful in the treatment of wounds and in the manufacture of certain eyewear.

See e.g., Skjak-Braek, G., et al. (1988) Chemistry, Biochemistry, Physical Properties, andApplic. Elsivier, New York; and Shigemasa & Minami (1996) Applications ofChitin and Chitosan for Biomolecules in Biotechnol Genet. Eng. Rev. 13:383.

Oligosaccharide components of chitin have been reported to be particularly useful. For example, specific chitin oligosaccharides have been reported to exhibit significant anti-cholesteremic, anti-bacterial, anti-fungal, immunomodulatory, or anti- tumor activity. Applications for certain chitin oligosaccharides or mixtures of thereof have been disclosed. Kendra, D.G. and Hadwiger, L.A. (1984) Experimental Mycology 8: 276; and Ryan, CA. (1994) Proc. Natl. Acad. Sci., USA 91: 1.

Methods for degrading chitin into oligosaccharides have attracted substantial interest. In general, the methods can be grouped into biological and synthetic methods.

For example, there have been efforts to identify biological methods for degrading chitin. Such efforts have focussed on identifying organisms capable of utilizing chitin as a carbon source. These organisms are sometimes referred to as being "chitinivorous." In particular, Aeromonas, Vibrio and certain other bacteria have been disclosed as utilizing chitin. See e.g., Roffey, P.E. and Pemberton, J.M. (1990) Current Microbiol 21:329; Jannatipour, M., et al. (1987) J. Bacteriol. 169:3785; and Soto-Gil, R.W. and Zyskind, J.W. (1989) J. Biol. Chem. 264: 14778.

There have been attempts to understand how chitinivorous organisms degrade chitin. For example, in one approach, recombinant DNA techniques have been used to identify genes that encode proteins that catabolize chitin. The approach has revealed a number of specific enzymes organized in pathways. Some of the enzymes are referred to as "chitinases". See e.g., Skjak-Braek, G., et al., supra, Flach, j., et al. (1992) Experientia. 48: 701; Robbins, P.W., et al. (1988) J. Biol. Chem. 263: 443 ; Fuche, et al., (1986) Appl. Environ. Microbiol. 51: 504; Kamei, K. et al. (1989) J. Biol. Chem. 105: 979-985; Watanabe, T., et al. (1992) J. Bacteriol. 1 A: 408; U.S. Pat. Nos. 5,352,607, 5,374,540, and 4,751,081; and EPO 01573.

Biochemical approaches have provided additional insight into the chitinases. See e.g., Colowick S.P. and Kaplan, N.O., (1988) et/j. in Enzymol. 161: 403; and Kuranda, M.J., and Robbins, P.W. (1991) J. Biol. Chem. 266: 19758. There has been some conflict in the field as to how chitinovirous organisms degrade chitin. For example, some reports disclose only two chitin degrading enzymes.

Significant attention has focussed on certain chitinivorous marine bacteria. For example, several important chitinolytic pathways have been identified in Vibrio strains and particularly Vibrio furnissii. Recombinant DNA techniques have been used in attempts to understand the chitinolytic pathways. See e.g., Bassler, B.L., et al. (1991) J. Biol. Chem. 266: 24276; Bassler, B.L., et al. (1991) J. Biol. Chem. 266: 24268; Yu, C, et al. (1991) J. Biol. Chem. 266: 24260; and references cited therein.

Further, practice of most prior recombinant DNA techniques has posed problems. For example, the techniques have not provided effective introduction of foreign DNA into prokaryotic cells such as marine bacteria and especially Vibrio strains such as Vibrio furnissii.

The prior biological methods have suffered from other shortcomings. For example, there has been recognition that insufficient attention has been focussed on identifying and analyzing enzymatic pathways in chitinivorous organisms. Additionally, some of the methods are reported to require significant effort to isolate chitin oligosaccharides. For example, in a specific prior method, extensive chromatographic manipulations were needed to resolve chitin oligosaccharides derived from chitinase treatment. See e.g, WO 97/31121; and references cited therein.

As noted, synthetic methods have also been used to degrade chitin. For example, there has been substantial attention focussed on developing effective approaches for making chitin oligosaccharides on a commercial scale. In a specific prior method, a large amount of chitin is hydro lyzed with a strong acid and resulting oligosaccharides are purified, e.g, by chromatography. See e.g., US. Pat. Nos, 4,804,750 and 5,705,634. There has been recognition that many synthetic methods are not always optimal for making chitin oligosaccharides. For example, many synthetic methods are not always cost effective particularly in settings where a large quantity of a specific chitin oligosaccharide is needed. In addition, the methods may not be desirable in situations where use of harsh chemicals such acids is to be avoided. Further, many prior synthetic methods are not useful for resolving complex mixtures of chitin oligosaccharides.

Accordingly, most prior biological and synthetic methods that attempt to degrade chitin into component oligosaccharides have not made the oligosaccharides readily available at minimal cost. Further, practice of the prior methods has not enhanced the purity of many chitin oligosaccharide preparations. Thus, in general, it has been difficult to make and use many types of chitin oligosaccharides.

It would be desirable to have chitinases and particularly extracellular (E-)- chitinases to convert chitin into useful oligosaccharide components. It would be particularly desirable to have efficient methods for using the E-chitinases to make specific chitin oligosaccharides or desired mixtures thereof at minimal cost and without significant use of harsh chemicals.

SUMMARY OF THE INVENTION

The present invention generally relates to chitin catabolizing enzymes and particularly to extracellular chitinases (E-chitinases) including recombinant E-chitinases and fragments and derivatives thereof. In one aspect, the invention provides isolated polynucleotides that encode the E-chitinases or functional fragments or derivatives thereof. Additionally provided are methods for using the polynucleotides and polypeptides to make specific chitin oligosaccharides or desired mixtures thereof. The invention also provides highly useful recombinant DNA methods for introducing polynucleotides into prokaryotic cells and particularly certain marine bacteria such as those disclosed below. We have discovered a novel chitin catabolizing protein and particularly an E- chitinase that is derived from a marine bacterium. The E-chitinase effectively hydrolyzes chitin into component oligosaccharides preferably consisting of between about one and three subunits. The E-chitinase or certain fragments of the E-chitinase can be manipulated in accord with the invention to produce a substantially pure preparation of the chitin oligosaccharides. Additionally, we have found that it is possible to use the E- chitinases to make specific mixtures of chitin oligosaccharides and to facilitate resolution of those mixtures with certain recombinant prokaryotic cells. Importantly, practice of the present invention can provide specific chitin oligosaccharides or desired mixtures thereof at minimal cost. Further, the invention can provide those chitin oligosaccharides economically on a large scale and without significant use of harsh chemicals.

The present invention provides a number of significant uses and advantages. For example, the invention provides, for the first time, a recombinant E-chitinase from the marine bacterium Vibrio furnissii. That bacterium is believed to have evolved especially efficient processes for seeking-out and utilizing chitin in oceanic environments. In particular, the present invention relates to Vibrio furnissii E-chitinase and functional fragments thereof that are capable of degrading chitin when present in minute amounts. Significantly, the E-chitinases of the present invention can function in brackish or even significantly saline environments, thereby providing efficient chitinolysis in settings where substantial amounts of NaCI or related salts are present. Accordingly, use of this invention can provide highly efficient chitin hydrolysis under many adverse reaction conditions.

More specifically, the present invention provides useful polynucleotides and polypeptides relating to the E-chitinase that can be used in a variety of industrial, medical, agricultural, home or research settings. For example, the polynucleotides and polypeptides disclosed herein can be used to facilitate the manufacture of a variety of chitin oligosaccharides or desired mixtures thereof. As discussed, chitin oligosaccharides are highly useful. In particular, purified or partially purified preparations of the E- chitinase (or functional fragments thereof) can be used to control or eradicate certain fungal and insect pests. For example, the E-chitinase of this invention can be used alone or in combination with recognized biocides to treat or prevent attack from these pests. See U.S. Pat. No. 5,173,419 for disclosure relating to methods for using chitinases to inhibit or eradicate fungal and insect pests.

Additionally, the polynucleotides and polypeptides of the present invention have a wide spectrum of further uses and advantages. For example, the molecules can be employed in functional, cellular and molecular assays (e.g., screens) and in structural analysis, including X-ray crystallography, nuclear magnetic resonance imaging (NMRI), and computational techniques. For example, certain polynucleotides of this invention can be used as mutagenic targets to modulate the E-chitinase particularly to enhance chitinolysis or to fine-tune enzymatic activity. Of particular interest are techniques involving computer-assisted simulation of E-chitinase to facilitate production of molecules with enhanced chitinolytic activity. If desired, the molecules of this invention can be detectably-labeled with a suitable tag such as a radionuclide or other suitable component to facilitate detection of the E-chitinase in laboratory or oceanic environments.

Polypeptides and polynucleotides of this invention providing or facilitating chitinolysis can be provided in kit form or other convenient form to promote the manufacture, packaging, dissemination, storage, and/or use of the present invention.

Particular molecules of the present invention provide further important uses and advantages. For example, the E-chitinase or functional fragments thereof provided by this invention can be used to degrade chitin and to make specific chitin oligosaccharides or desired mixtures thereof such as GlcNac as well as oligomers consisting of between about two and three substituents. Significantly, the methods of the present invention can be used to make chitin oligosaccharides in forms suitable for many applications such as those relating to research, medical, agricultural or commercial use. As will be discussed in more detail below, the invention is particularly useful for making small chitin oligosaccharides, i.e., chitin oligosaccharides having from between about 1 and 3 covalently linked GlcNAc substituents.

In addition, use of the present E-chitinases as well as functional fragments or derivatives thereof is enhanced by the recombinant DNA techniques discussed below. More specifically, the recombinant DNA techniques can be used to enhance introduction of polynucleotides into certain bacteria and particularly Vibrio strains such as Vibrio furnissii.

Accordingly, in one aspect, the present invention provides an isolated polynucleotide that encode&.an E-chitinase, particularly a Vibrio E-chitinase and more particularly a Vibrio furnissii E-chitinase. Also provided are functional fragments and derivatives of the E-chitinase. In one embodiment, the polynucleotide encodes a Vibrio furnissii E-chitinase that is capable of hydrolyzing chitin into small chitin oligosaccharides as determined by a suitable chitinase assay. In another embodiment, the invention provides an isolated polynucleotide that encodes a fragment or a derivative of the E-chitinase that is capable of hydrolyzing chitin into small chitin oligosaccharides as determined in the chitinase assay.

The chitin hydrolysis provided by the E-chitinases and functional fragments thereof can be monitored and quantitated, if desired, by one or a combination of strategies. For example, a preferred chitinase assay involves detection of GlcNAc and small chitin oligosaccharides having the formula [(GlcNAc)_n ], wherein n is between from about 1 to about 3 and typically about 2.

In more particular embodiments, the polynucleotide is DNA that encodes a polypeptide capable of binding and preferably hydrolyzing chitin as determined by assays described herein. In a specific embodiment, the DNA encodes a polypeptide that is capable of hydrolyzing from between about 80% up to about 99% (w/w) or greater of the total amount of chitin present in the assay. More preferred are those polypeptides that can produce at least from about 70% up to about 90% (mole percent) of GlcNAc, (GlcNac)₂, (GlcNac)₃ or a mixture thereof relative to the mole amount of chitin in the assay. Illustrative methods for identifying such polynucleotides and characterizing the encoded polypeptides are provided below.

Additionally preferred polynucleotides of this invention are capable of hybridizing to the nucleotide sequence shown in Figure 3 (SEQ LD NO. 1) or the complement thereof under at least moderate stringency hybridization conditions (ie. moderate or high stringency conditions). In a preferred embodiment, the polynucleotide will also hybridize to the nucleotide sequence shown in Figure 3 (SEQ ID NO. 1) under high stringency conditions. The terms "moderate" and "high" hybridization stringency will have meaning to those of skill in this field. See the examples and discussion that follows for more specific disclosure relating to moderate and high stringency hybridization conditions.

In one embodiment, the polynucleotide is capable of hybridizing to the nucleotide sequence shown in Figure 3 (SEQ ID NO. 1) or the complement thereof under high stringency conditions and is preferably between from about 12 to about 50 nucleotides in length. Illustrative of such polynucleotides are oligonucleotide primers made by conventional synthetic methods. In another embodiment, the polynucleotide is between from about 60 to about 100 nucleotides in length up to about 3500 nucleotides in length or greater. Illustrative of such polynucleotides are restriction enzyme fragments or chemically synthesized fragments that are complementary to the nucleotide sequences shown in Figure 3 (SEQ ID NO. 1).

Additionally preferred polynucleotides of this invention encode a polypeptide and particularly a Vibrio furnissii E-chitinase having a molecular weight of between from about 60 kDa to about 100 kDa as determined by conventional protein sizing techniques such as polyacrylamide gel electrophoresis using sodium dodecyl sulfate (SDS) as described below.

Further preferred polynucleotides of this invention have at least about 70 percent sequence identity to the nucleotide sequence shown in Figure 3 (SEQ ID NO.l) or the complement thereof. Such sequence similarity (i.e. about 70% or greater) will sometimes be referred to herein as "substantial homology" or like term.

Additionally preferred polynucleotides of this invention encode an amino acid sequence that includes at least a chitinase domain of the Vibrio furnissii E-chitinase shown in Figure 4 (SEQ ID NO. 2). By the term "chitinase domain" or related term is meant that portion of the E-chitinase sequence shown in Figure 4 (SEQ ID NO. 2) that encodes a sequence with capacity to hydrolyze and preferably bind chitin or pNP- (GlcNAc) as determined by a standard chitinase assay. Especially preferred chitinase domains are sometimes referred to herein as "functional E-chitinase fragments" or like term which fragments preferably exhibit at least about 60% and more preferably at least about 70%, 80%, 90% or 95% up to about 99% or more of the chitinase activity of the E- chitinase amino acid sequence shown in Figure 4 (SEQ ID NO. 2) as determined in the standard chitinase assay. Exemplary methods for identifying chitinase domains are discussed more fully below.

Sometimes the E-chitinase sequence shown in Figure 3 (SEQ ID NO. 1) or the complement thereof will be referred to as "full-length" or like term to denote encoding by the Vibrio furnissii chiE gene.

Specifically preferred polynucleotides of this invention encode the full-length Vibrio furnissii E-chitinase shown in Figure 4 (SEQ ID NO. 2). Also specifically preferred are fragments and derivatives thereof including functional fragments of the E- chitinase sequence. Additionally provided by the present invention are recombinant vectors that can include at least one isolated polynucleotide of this invention. In some embodiments, it may be useful to provide multiple copies of the E-chitinase sequence shown in Figure 3 (SEQ ID NO. 1) up to about 2 to about 5 of such copies including functional fragments or derivatives of the polynucleotides. The multiple copies can be provided in contiguous or non-contiguous formats as desired. It is generally preferred that the recombinant vector be capable of propagating the isolated polynucleotide in a suitable cell such as a prokaryotic host cell. However, in some cases, it may be useful to use a eukaryotic host cell ( e.g., insect, yeast or fungal cell) to propagate the vector. Additionally preferred recombinant vectors are capable of expressing the isolated polynucleotide as RNA and usually mRNA, in the host cells. The recombinant vector can include nearly any number of useful elements as described more fully below. Illustrative recombinant vectors are provided below.

As noted, specifically preferred polynucleotides of the invention encode a Vibrio furnissii E-chitinase or a fragment or derivative thereof. In one embodiment, the polynucleotides are substantially homologous to the E-chitinase sequence shown in Figure 3 (SEQ ID NO. 1). Preferred are polynucleotides including and more preferably consisting of DNA having a length of between from about 50 up to about 3000 nucleotides or more and most preferably about 2550 to 2560 nucleotides, as determined by standard nucleic acid sizing methods. In another embodiment, the isolated polynucleotide includes and more preferably consists of RNA and particularly mRNA that is complementary to the Vibrio furnissii E-chitinase sequence shown in Figure 3 (SEQ ID NO. 1). The mRNA can be of the same or similar length as the DNA.

Also provided are host cells that include at least one polynucleotide of this invention including a fragment or derivative of that polynucleotide. Preferably, the host cells are maintained under suitable cell culture conditions to express the desired amino acid sequence in the host cell, including the host cell periplasm; cell medium, or both. The invention also includes methods for isolating a polynucleotide of this invention. In one embodiment, the methods including isolating the full-length E- chitinase sequence disclosed in Figure 3 (SEQ ID NO. 1) or the complement thereof. In general, the methods include introducing the polynucleotide into host cells, typically as a recombinant vector including one or more copies of the polynucleotide, culturing the host cells under conditions suitable for propagating the polynucleotide and purifying the polynucleotide from the host cells. Host cells useful for propagating the polynucleotides will typically be prokaryotic cells although certain eukaryotic host cells may be preferred in some instances. Preferred are prokaryotic cells of the genus Vibrio and particularly Vibrio furnissii and related strains. Additionally preferred prokaryotic host cells include E. coli and certain Bacillus strains useful for making DNA. Especially preferred are host cells that can be adapted to produce the polynucleotides and polypeptides of this invention on a commercial scale such as E. coli. Alternatively, the Polymerase Chain Reaction (PCR) amplification or related nucleic acid amplification methods can be used to isolate significant quantities of the polynucleotides provided herein.

Further provided are cultured host cells that have been transformed, transfected or infected either transiently or stably by at least one type of recombinant vector of this invention. In a preferred embodiment, the host cells include a recombinant vector which comprises an isolated polynucleotide that encodes the Vibrio furnissii E-chitinase or a functional fragment or derivative thereof.

The present invention also provides useful oligonucleotide primers, typically single-stranded primers, that are complementary to the polynucleotides provided herein. In one embodiment, the oligonucleotide primers are complementary to the Vibrio furnissii E-chitinase sequence shown in Figure 3 (SEQ ID NO. 1) or the complement thereof. The oligonucleotide primers have a variety of useful applications, e.g., to detect or amplify a desired polynucleotide of this invention including the full-length E-chitinase. Exemplary oligonucleotide primers will generally have length of between about 12 to about 70 nucleotides, preferably between about 15 to about 25 or 30 nucleotides, typically about 20 nucleotides although somewhat larger or smaller primers are useful for some applications.

In another aspect of the present invention there is provided isolated polypeptides that are capable of binding and preferably also hydrolyzing chitin as determined in chitin binding and chitinase assays described below. In one embodiment, the polypeptide is capable of hydrolyzing from between about 60% up to about 99% (w/w) or greater of the total amount of chitin present in the assay as discussed above. In a particularly preferred embodiment, the polypeptide is the Vibrio furnissii E-chitinase illustrated in Figure 4 (SEQ ID NO. 2).

Additionally preferred polypeptides are isolated E-chitinases, preferably Vibrio E- chitinases and more preferably Vibrio furnissii E-chitinases or functional fragments or derivatives thereof having an apparent molecular weight of between about 60 to about 100 kDa. The molecular weight of the polypeptide can be determined by a variety of standard means including polyacrylamide gel electrophoresis. Preferred are polypeptides having between about 100 to about 1000 amino acids and more preferably between about 500 to about 900 amino acids. Specifically preferred is the Vibrio furnissii E-chitinase sequence shown in Figure 4 (SEQ ID NO. 2).

Additionally preferred are isolated polypeptides that exhibit at least about 70 percent amino acid identity to the polypeptide sequence illustrated in Figure 4 (SEQ ID NO. 2). Additionally preferred are functional fragments or derivatives of that polypeptide.

Further provided is a substantially pure enzyme preparation that includes at least one of: 1) the isolated E-chitinase shown in Figure 4 (SEQ ID NO. 2), 2) a functional fragment of the E-chitinase, or 3) a derivative of the isolated E-chitinase. Also contemplated are substantially pure enzyme preparations that include at least one other Vibrio furnissii chitinolytic enzyme as provided below. The invention also provides methods for producing an isolated E-chitinase of this invention including functional fragments thereof which methods generally include culturing suitable host cells comprising a polynucleotide expressing the E-chitinase or functional E-chitinase fragment. Preferred are culture conditions using medium suitable for expressing the E-chitinase or functional fragment in the host cell or medium.

Isolated molecules of this invention can be obtained as a substantially pure preparation if desired. For example, the polynucleotides, polypeptides, and chitin oligosaccharides disclosed herein can be isolated in substantially pure form by standard methods and can be provided as sterile preparations if desired. Specifically preferred methods for providing substantially pure preparations of the polynucleotides and polypeptides are discussed below.

In another aspect, the present invention provides methods for producing a substantially pure preparation of a specific chitin oligosaccharide or mixture of specific chitin oligosaccharides.

In one embodiment, there is provided a method for making N, N' diacetylchitobiose [(GlcNAc) ]. In one embodiment, the method includes at least one and preferably all of the following steps: a) contacting a sufficient amount of chitin with an E-chitinase under conditions sufficient to form an oligosaccharide preparation comprising N-acetyl-D- glucosamine (GlcNAc), N, N'-diacetylchitobiose [(GlcNAc)₂ ], and a GlcNAc trisaccharide [(GlcNAc)₃ ], b) removing substantially all of the N-acetyl-D-glucosamine (GlcNAc) and the GlcNAc trisaccharide [(GlcNAc)₃ ] from the preparation; and c) producing the substantially pure preparation of the N, N'-diacetylchitobiose [(GlcNAc)₂ ] from the preparation. The E-chitinase is preferably a Vibrio E-chitinase and more preferably the Vibrio furnissii E-chitinase illustrated in Figure 4 (SEQ ID NO. 2). However, in other embodiments of the method one or more functional fragments or derivatives of the E- chitinase may be used if desired. The E-chitinase or functional fragment used in the method can be provided from a variety of sources. For example, the enzyme can be purified or partially purified from certain host cells such as Vibrio furnissii cells using conventional protein purification techniques discussed below. Preferably, the Vibrio furnissii E-chitinase is provided as a substantially pure enzyme preparation.

In a preferred embodiment of the method, the Vibrio furnissii E-chitinase is provided by recombinant prokaryotic cells that carry a polynucleotide encoding the E- chitinase. Use of the recombinant prokaryotic cells has advantages which will be apparent to those of skill in this field. For example, by using the recombinant prokaryotic cells, a renewable source of E-chitinase is provided. Preferred prokaryotic cells are capable of secreting the E-chitinase into medium or buffer. Specifically preferred are the recombinant E. coli strains described below. Functional fragments of the E-chitinase or derivatives thereof may also be employed, e.g., in host cells where enhanced secretion of expressed protein is desired.

As noted, it is an object of the present invention to provide effective methods for resolving complex mixtures of chitin oligosaccharides. Prior practice has attempted to achieve this objective by employing difficult chromatographic approaches. As discussed, such approaches are not always optimal. The present invention achieves effective resolution of the oligosaccharides by using prokaroytic cells which can selectively catabolize certain chitin oligosaccharides. Thus, the need to use difficult separation techniques is reduced and preferably avoided.

For example, preferred use of the method for producing the N, N¹ diacetylchitobiose [(GlcNac)₂ ] (see above) includes substantially removing the N-acetyl- D-glucosamine (GlcNAc) from the oligosaccharide preparation. Typically, the removal is accomplished by exposing the preparation to prokaryotic cells capable of consuming the N-acetyl-D-glucosamine (GlcNAc). In some instances, it will be desirable to provide conditions conducive to the survival and preferably propagation of the prokaryotic cells. The prokaryotic cells can be added at any suitable step in the method although step b) above, is generally preferred for most applications.

The prokaryotic cells used in the method include and preferably consist of an E. coli strain capable of selectively utilizing the N-acetyl-D-glucosamine (GlcNAc). Preferably, the E. coli strain is substantially or completely incapable of effectively utilizing the chitin disaccharide. Illustrative of such E. coli strains is the strain designated Xm.1.4 described in detail below.

The present methods for producing chitin oligosaccharides are flexible and can be readily adapted to suit intended use. For example, in one embodiment of the method for producing the substantially pure preparation of N, N' diacetylchitobiose [(GlcNac)₂ ], the method further includes deproteinizing the oligosaccharide preparation. Deproteinization is desirable for several reasons including facilitating oligosaccharide purification. The deproteinization step can be conducted at nearly any step in the method although step a) will usually be preferred. In this embodiment, the method can optionally include contacting the oligosaccharide preparation (deproteinized) with a suitable ion exchange resin. Typically, the contacting will be performed at step b) of the method, although the contacting can be conducted at another step if desired. In a further embodiment of the method, step b) can include crystallization of the N, N'-diacetylchitobiose [(GlcNAc)₂] from the oligosaccharide preparation by adding a lower alcohol thereto. The lower alcohol employed typically will include from about 1 to about 3 carbons such as methanol.

In another embodiment of the method for producing the substantially pure preparation of N, N' diacetylchitobiose, the method can further include contacting the oligosaccharide preparation formed in step a) with a Vibrio furnissii periplasmic β- acetylglucosaminidase (Exo I) under conditions capable of hydrolyzing the GlcNAc trisaccharide [(GlcNAc)₃ ]. Contact of the oligosaccharide preparation can be conducted at any step of the method although step b) will generally be preferred. The Exol can be provided as part of a substantially pure enzyme preparation. Alternatively, the Exol can be provided by adding suitable recombinant prokaryotic cells that have been transformed to express the Exo I. Specific prokaryotic host cell strains, e.g., E. coli, are specifically provided below.

In another aspect, the present invention also provides a kit that includes a container means comprising at least one of: 1) an isolated polynucleotide comprising sequence with at least about 70% sequence identity to the sequence shown in Figure 3 (SEQ ID NO. 1) or a functional fragment or a derivative of that sequence; 2) a pair of oligonucleotide primers capable of hybridizing to the sequence shown in Figure 3 (SEQ ID NO. 1) or the complement thereof preferably under high stringency conditions; 3) a polypeptide with at least about 70% sequence identity to the sequence shown in Figure 4 (SEQ ID NO. 2) or a fragment or a derivative thereof; and 4) a bacterial cell culture capable of resolving a mixture of chitin oligosaccharides. Preferably, the bacterial cell culture is capable of removing a desired chitin monosaccharide and/or oligosaccharides from a mixture of oligosaccharides. A specifically preferred strain is the E. coli strain Xm.1.4.

In preferred embodiments, the kit is formatted to provide an efficient system for degrading chitin and particularly for producing a desired chitin oligosaccharide or desired mixtures thereof. In one embodiment, the kit will provide sufficient components for commercial scale preparation of the chitin oligosaccharide while in another embodiment, the kit will provide enough components for smaller scale preparation of the oligosaccharides such as those usually encountered in home, research or medical use. Preferred kits are particularly well-suited for making [(GlcNac)₂ ]. In another aspect, the present invention provides useful recombinant DNA methods for introducing foreign DNA into prokaryotic cells including marine bacteria such as certain Vibrio strains. As disclosed below, the recombinant DNA methods are especially useful for enhancing introduction of the foreign DNA into cells that include a restriction-modification (R-M) system.

In general, most R-M systems protect cells from intrusion by foreign DNA. Protection is accomplished by expression of a DNA restriction endonuclease and its corresponding DNA modification enzyme. The DNA restriction endonuclease specifically cleaves the foreign DNA while the DNA modification enzyme protects the DNA from the endonuclease. The present methods minimize and preferably eliminate effects of the R-M system by protectively modifying a foreign DNA of interest prior to introduction into prokaryotic cells. More particularly, the present methods provide for cell-mediated protective modification of foreign DNA and, optionally, introduction of the modified foreign DNA into the prokaryotic cells. The prokaryotic cell may have a demonstrable R-M system or only may be suspected of having an R-M system (or more than one R-M system). Introduction of the protectively modified DNA can be achieved by one or a combination of different strategies such as those mentioned below.

Accordingly, in one embodiment, there is provided a method for transforming prokaryotic cells comprising an R-M system in which the method includes at least one of the following steps and preferably all of the following steps: a) introducing foreign DNA, e.g., a recombinant vector, into first prokaryotic cells which cells include a recombinant DNA sequence comprising a DNA segment encoding a prokaryotic DNA methylase, b) maintaining the first prokaryotic cells under conditions sufficient to methylate the foreign DNA in the cells, c) isolating the methylated foreign DNA from the first prokaryotic cells; and d) introducing the isolated and methylated foreign DNA into second prokaryotic cells comprising the R-M system under conditions sufficient to transform the second prokaryotic cells with the isolated and methylated foreign DNA.

In a preferred embodiment of the method, prior to step a), the recombinant sequence is isolated by conventional DNA methylase selection and the first prokaryotic cells are transformed with the recombinant sequence under conditions capable of expressing the DNA methylase gene in the first prokaryotic cells. Preferred methods for conducting DNA methylase selection are discussed below. In a specific embodiment, the isolated and methylated foreign DNA and particularly an isolated and methylated recombinant vector is resistant to cleavage by restriction endonuclease Sau96I or an isoschizomer thereof as determined, e.g., by a standard DNA restriction enzyme assay. Preferred are isoschizomers of Sau96I endogenous to the R-M system of Vibrio furnissii.

As will become more apparent from the discussion and examples below, the present methods for transforming foreign DNA are generally applicable and are not limited to a specific type of cell or DNA methylase. In particular, the methods can be used to transform a variety of foreign DNAs into prokaryotic cells that have or are suspected of having an R-M system.

For example, the first prokaryotic cells can be nearly any bacterial cell capable of expressing a DNA methylase and particularly a heterospecific bacterial DNA methylase. Illustrative first prokaryotic cells include E. coli and certain other bacteria that can be manipulated in accord with standard recombinant DNA techniques to express a heterospecific bacterial DNA methylase at levels sufficient to protectively modify (ie. methylate) pj hemimethylate a foreign DNA sequence of interest. The recombinant sequence can be introduced into the first prokaryotic cells, e.g., by transformation, and can encode one or several heterospecific bacterial DNA methylases. Choice of the heterospecific bacterial DNA methylase will be guided by several parameters including the R-M system encountered. Particularly preferred are DNA methylases isolated from Vibrio cells and especially Vibrio furnissii cells according to methods disclosed herein.

As noted, a protectively modified foreign DNA is resistant to cleavage by the R- M system of the prokaryotic cells. That resistance can be measured by a variety of conventional methods such as by a standard restriction enzyme cleavage assay. Preferred heterospecific bacterial DNA methylases are capable of reducing site-specific cleavage of isolated and methylated foreign DNA by about 95% to about 99% or more when compared to a suitable control sequence such as unmethylated pACYC185. Additionally preferred are heterospecific bacterial DNA methylases that do not significantly reduce replication efficiency of the recombinant vector in the second prokaryotic cells, ie. not more than about 1% to about 5% as determined in a standard DNA replication assay.

It is recognized that some bacterial cells have one R-M system; whereas others have several R-M systems, each acting essentially independently at a different DNA sequence. Preferred use of the present methods involves use of second prokaryotic cells comprising only a few R-M systems, generally less than 2 or 3 R-M systems, and preferably one R-M system manifesting a recognized restriction enzyme activity. More specifically, preferred second prokaryotic cells express only DNA restriction enzyme whose DNA recognition site(s) are known or can be readily ascertained by established methods e.g., by a standard restriction enzyme cleavage assay. More preferred second prokaryotic cells will comprise one R-M system with capacity to specifically cleave less than about 0.5% to about 5% of the foreign DNA protectively modified by the first prokaryotic cells as determined by the standard DNA restriction enzyme essay.

Especially preferred second prokaryotic cells for use in the method are marine bacteria such as certain Vibrio strains and particularly Vibrio furnissii cells that express an R-M system that includes the Sau96I isoschizomer. In another aspect, the present invention provides methods for transconjugating foreign DNA from suitable donor cells into recipient cells comprising an R-M system. In one embodiment, the method includes at least one and preferably all of the following steps: a) introducing foreign DNA, e.g., a recombinant vector, into first prokaryotic cells (donor cells) comprising a recombinant sequence comprising sequence encoding a DNA methylase gene, b) maintaining the donor cells under conditions sufficient to methylate the foreign

DNA in the cells, c) contacting the donor cells with second prokaryotic cells (recipient cells) under conditions conducive to transconjugation between the donor and recipient cells; and d) transconjugating the foreign DNA from the donor cells to the recipient cells.

In a preferred embodiment of the method, prior to step a), the recombinant sequence is isolated by conventional DNA methylase selection and the first prokaryotic cells are transformed with the recombinant sequence under conditions capable of expressing the DNA methylase gene in the first prokaryotic cells. Illustrative first and second prokaryotic cells include E. coli and Vibrio strains, respectively. In another embodiment, the methylated foreign DNA is resistant to cleavage by restriction endonuclease Sau96Ioτ an isoschizomer thereof as determined, e.g., by a standard DNA restriction enzyme assay. Preferred are isoschizomers of Sau96I endogenous to the R-M system of Vibrio furnissii. In this embodiment, the method provides several advantages including reducing or eliminating need to isolate methylated foreign DNA and higher transformation frequencies.

Further provided by the present invention are recombinant prokaryotic cells such as E. coli cells that include a Vibrio furnissii DNA methylase gene or suitable (functional) fragment thereof. Preferred are E. coli cells comprising a Vibrio furnissii DNA methylase gene or suitable fragment thereof that encodes a protein that can reduce specific cleavage of a test DNA sequence by its corresponding restriction enzyme from about 95% to about 99.5% or greater as determined, e.g., in a standard restriction enzyme cleavage assay. A particularly preferred E. coli strain has the designation AFIOIM and is described in detail below.

In another aspect, the present invention provides a library that includes a plurality of the polynucleotides or the polypeptides of this invention including fragments or derivatives of those polynucleotides or polypeptides. Illustrative of such libraries include cDNA and genomic DNA libraries, combinatorial and peptide expression libraries.

BRIEF DISCUSSION OF THE DRAWINGS

Figure 1 is a diagram outlining some aspects of chitin degradation by V. furnissii. Chitin outside the cell (out) is hydrolyzed by the E-chitinase of the present invention (1) to form various oligomers of N-acetyl-D-glucosamine (GlcNAc). The oligomers are denoted as G • The G N is modified by certain periplasmic proteins to form GlcNAc and (GlcNAc)₂ ( G and G₂ , respectively). The G and G₂ are further catabolized inside the cell (in).

Figure 2 is a drawing outlining the molecular cloning of the V. furnissii E- chitinase gene.

Figure 3 is a drawing showing the nucleotide sequence (SEQ ID NO. 1) of V. furnissii extracellular (E-) chitinase.

Figure 4 is a drawing showing the predicted amino acid sequence (SEQ ID NO. 2) of the V. furnissii extracellular (E-) chitinase sequence shown in Figure 3.

Figures 5 A-5B are a schematic diagrams outlining procedures for constructing V. furnissii E-chitinase null mutants. (5 A) construction of PNQ-chiE-sacB and use for transformation and transconjugation. (5B) construction of pSF/chiE and use for transformation and transconjugation.

Figure 6 is a drawing showing the cloning and expression of Vibrio furnissii DNA methylase.

DETAILED DECRIPTION OF THE INVENTION

As summarized above, the present invention relates, in one aspect, to recombinant E-chitinases and fragments and derivatives thereof. The invention also provides isolated polynucleotides that encode the E-chitinase and fragments or derivatives of the E- chitinase. Further provided are polypeptides encoded by the polynucleotides. Additionally provided are methods for making various chitin oligosaccharides using the polynucleotides and polypeptides provided by this invention.

In general, optimal practice of the present invention can be achieved by use of recognized laboratory manipulations. For example, techniques for purifying nucleic acids, methods for making and screening DNA libraries, methods for making recombinant vectors, cleaving DNA with restriction enzymes, ligating DNA, introducing DNA into host cells by stable or transient means, culturing the host cells, methods for isolating and purifying polypeptides, methods for making certain chitin oligosaccharides, and computer-assisted methods for detecting nucleic acid or amino acid sequence homology have been reported. See generally Sambrook et al., Molecular Cloning (2d ed. 1989); Ausubel et al., Current Protocols in Molecular Biology, (1989) John Wiley & Sons, New York; S. Altschul et al. (1997) Nuc. Acids Res., 25: 3389.

Additionally, more specific laboratory methods have been optimized for use with V. furnissii. Such methods include, but are not limited to, methods for making chitin, chitosan, certain oligosaccharides of chitin or chitosan, and related techniques. See e.g., Yu, C, et al. (1987) Biochem. Biophys. Res. Comm. 149: 86; Yu, C, et al. (1991) J. Biol. Chem. 266: 24260; Bouma, C. and Roseman, S. (1996) J. Biol. Chem. 271 : 33457; Bouma, C. and Roseman, S. (1996) J. Biol. Chem. 271: 33468; Chitlaru, E. and Roseman, S. (1996) J. Biol Chem. 271: 33433; Keyhani, N.O. and Roseman, S. (1996) J. Biol. Chem. 271 : 33425; Wang, L.X., et al. (1997) Glycobiology 1: 855; Horowitz, et al. (1957) J. Am. Chem. Soc. 79: 5046; Roseman, S. and Daffner, I. (1956) Anal. Chem. 28: 1743; Johnston, I., et al. (1966) J. Biol. Chem. 241: 5735; Roseman, S. and Ludowieg, J. (1954) J. Am. Chem. Soc. 76: 301.

Polynucleotides encoding specific β-N-acetylglucosaminidases from Vibrio furnissii have been disclosed. Some of the disclosed enzymes are represented schematically in Figure 1. The polynucleotides include endl (periplasmic chitodextrinase (Endo-I) ), exol (periplasmic β-GlcNAcidase (Exo-I) ) and exoll (an enzyme, aryl β-N-acetylglucosaminidase, specific for aryl β-N-acetylglucosaminides (Exo-II) ). Further disclosed are specific host strains comprising the polynucleotides, mutant strains of Vibrio furnissii lacking part or all of the disclosed polynucleotides, as well as methods for making certain chitin oligosaccharides using disclosed enzymes, transformed host strains, and mutant V furnissii strains. See U.S. Patent No.5,792,647; pending U.S. Application No. 08/600,452, filed on February 13, 1996; and published PCT Application No. WO 96/25424, filed on February 13, 1996; the teachings of which are fully incorporated herein by reference.

As discussed, the present invention generally relates to polynucleotides and polypeptides associated with chitinolysis in certain Vibrio strains and particularly Vibrio furnissii. More particularly, the present invention features polynucleotides and polypeptides encoded by same that are capable of hydrolyzing chitin. The present invention more specifically relates to the Vibrio furnissii E-chitinase gene and enzymatically active protein fragments encoded by the gene. See Figures 3 and 4 (SEQ ID NO. 1 and 2, respectively). The E-chitinases of this invention are similar to exoenzymes. That is, in most instances the E-chitinases will usually yield disaccharides particularly at the non-reducing end of the linear polymer. Polynucleotides of this invention (DNA, including genomic DNA; RNA, mRNA or chimeras thereof) can be derived from a variety of sources, preferably from marine bacteria of the genus Vibrio and particularly Vibrio furnissii. It will be appreciated that the present disclosure provides ample information to facilitate isolation of a variety of E- chitinases, including isoforms (isozymes or isoenzymes) thereof, through use of recognized molecular techniques such as PCR and related amplification techniques. Typically, the isolated polynucleotide will be subcloned into a suitable recombinant vector, although in some cases it may be desirable to make and use the polynucleotide without a recombinant vector, e.g., as a PCR-amplified product.

In most instances, the polynucleotide is provided in a suitable recombinant DNA vector capable for expressing the encoded E-chitinase (or fragment or derivative thereof) in a cell expression system, e.g., a prokaryotic cell expression system. The polynucleotide may include operably linked transcriptional elements as needed such as a promoter, leader sequences to help drive expression of the encoded polypeptide in a desired host cell expression system. Alternatively, the recombinant DNA vector itself may provide some or all of the control elements. In general, polynucleotides of the invention are often made so that naturally-occurring E-chitinase control sequences (e.g., genomic control sequences) are reduced in number and preferably removed.

As discussed, the present invention provides isolated polynucleotides that preferably encode Vibrio furnissii E-chitinase or a fragment or a derivative thereof. A specifically preferred polynucleotide is that shown in Figure 3 (SEQ ID NO. 1). The isolated polynucleotides may be cloned or subcloned using nearly any method known in the art. See e.g., Sambrook, J. et al., supra. In particular, nucleotide sequences of the invention may be cloned into any of a large variety of recombinant DNA vectors. Possible vectors include, but are not limited to, cosmids, plasmids or modified bacteriophages, although the vector system must be compatible with the host cell used. See e.g, Miller et al., Biotechniques, 7:980-990 (1984)), incoφorated herein by reference). Plasmids include, but are not limited to, pBR, PUC, and Bluescriptθ (Stratagene) plasmid derivatives. Introduction into and expression in host cells is done for example by, transformation, transfection, infection or electroporation with transconjugation being the method of choice for most applications, etc. Preferred methods for performing transformation and transconjugation in V. furnissii are detailed below.

The term "vector" or "recombinant vector" as used herein means any nucleic acid sequence of interest capable of being incorporated into a host cell and resulting in the expression of a nucleic acid sequence of interest. Vectors can include, e.g., linear nucleic acid sequences, plasmids, cosmids, phagemids, and extrachromosomal DNA. Specifically, the vector can be a recombinant DNA. Also used herein, the term "expression" or "gene expression", is meant to refer to the production of the protein product of the nucleic acid sequence of interest, including transcription of the DNA and translation of the RNA transcript. Most recombinant vectors will include a "cloning site" which as used herein is intended to encompass at least one restriction endonuclease site. Typically, multiple different restriction endonuclease sites (e.g., a polylinker) are contained within the vector to facilitate cloning.

As noted, preferred polynucleotides of this invention encode an E-chitinase having a molecular from between about 60, 70, 80, 90, to about 100 kDA. Also preferred are those polynucleotides that have at least about 70%, 75%, 80%, 90%, 95%, 99% or greater sequence identity to the nucleotide sequence shown in Figure 3 (SEQ ID NO. 1) or the complement of that nucleotide sequence. As will be fully appreciated, such sequences are substantially homologous to the sequences shown in SEQ LD NO. 1 or the complement thereof. A more preferred polynucleotide of the invention encodes the V. furnissii E- chitinase shown in Figure 4 and SEQ ID NO. 2. A specifically preferred polynucleotide is the V. furnissii E-chitinase sequence shown in SEQ ID NO. 1 or the complement thereof. By the term "substantially homologous" is meant relationship between two molecules and generally refers to subunit sequence similarity between the two molecules. Typically, the two molecules will be a DNA or protein sequence. For example, when a subunit position in two molecules is occupied by the same monomeric subunit, i.e. a nucleotide or an amino acid residue, then they are homologous at that position.

Homology between the two sequences is a direct function of the number of matching or homologous positions, e.g., if 50% of the subunit positions in the two DNA sequences are homologous then the two sequences are 50% homologous. By "substantially homologous" is meant largely but not wholly homologous. More particularly, the term is meant to denote at least about 70% or greater homology as defined above with respect to the V. furnissii E-chitinase sequence illustrated in Figure 3 (SEQ ID NO. 1) or the complement thereof.

Two substantially homologous polynucleotide sequences can be identified by one or a combination of different strategies. For example, in one approach, a polynucleotide of this invention that is substantially homologous to the sequence shown in Figure 3 (SEQ ID NO. 1) can be identified by employing moderately or highly stringent conditions. In particular, moderate stringency conditions are meant to include a hybridization buffer comprising about 20% formamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing once with that SSC buffer at 37°C. Additionally, highly stringent conditions are meant to include a hybridization buffer comprising about 50% formamide in SSC buffer at about 42 °C and remaining bound when washed in SSC buffer. See e.g., Sambrook et al. supra.

Additional methods of detecting and quantitating substantial homology include so-called "dry" methods and include use of publicly available computer programs that can readily determine homology between two nucleic acids or polypeptide sequences of known or partially known sequence. Exemplary of such programs include the BLAST and TFASTA programs available from the National Library of Medicine (Genbank). See also S. Altschul et al. (1997), supra, for specific disclosure relating to use of the BLAST program.

Nucleic acid fragments and derivatives of this invention preferably should comprise at least about 12 to about 50 nucleotides, at least about 60, 100 to 200 nucleotides, at least about 300, 400, to about 500 nucleotides, or at least about 1000, 1500, 2000, or 2500 nucleotides up to about 2550 nucleotides or greater. In some preferred embodiments, a nucleic acid derivative is bound to a suitable moiety, sometimes called a tag, which permits ready identification such as a radionucleotide, fluorescent or other chemical identifier. Preferred are functional E-chitinase fragments that are capable of degrading chitin as determined in the standard chitinase assay.

The polynucleotide sequences of the invention can be altered by mutations such as substitutions, additions or deletions (contiguous or non-contiguous) that can provide for substantially homologous nucleic acid sequences. In particular, a given nucleotide sequence can be mutated in vitro or in vivo, to create variations in the nucleotides, e.g., to form new or additional restriction endonuclease sites or to destroy preexisting ones and thereby to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used including, but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., J. Biol. Chem., 253:6551 (1978)), use of TAB Registered TM linkers (Pharmacia), PCR-directed mutagenesis, and the like.

It will be appreciated that due to the degeneracy of genetic code , a number of different nucleic acid sequences may be used in the practice of the present invention. This includes the substitution of different codons encoding the same amino acid residue within the sequence, thus producing a silent or nearly silent change. Almost every amino acid except tryptophan and methionine is represented by several codons. Often the base in the third position of a codon is not significant, because those amino acids having 4 different codons differ only in the third base. This feature, together with a tendency for similar amino acids to be represented by related codons, increases the probability that a single, random base change will result in no amino acid substitution or in one involving an amino acid of similar character. See generally Alberts et al., Molecular Biology of the Cell, (1989) Garland Publishing, New York.

Reference herein to a "functional fragment" of E-chitinase or related term means a a preferred portion of the polypeptide shown in Figure 4 (SEQ ID NO. 2). As discussed, preferred functional fragments have capacity to hydrolyze and preferably bind chitin or pNP-(GlcNAc)₂ as determined by assays described herein. Additionally contemplated are polynucleotides that encode the functional fragment.

Functional fragments of E-chitinases of this invention can be made by one or a combination of conventional strategies. For example, in one approach, recognized recombinant methods can be used to create the fragments. In particular, the E-chitinase fragments can be targeted N- or C-terminal deletions (contiguous or non-contiguous) of a desired length, e.g., 1, 2, 3, 4, or 5 amino acids in length. If desired, larger deletions can be made up to about 10 to about 20 amino acids in length or more. In a more specific approach, site-directed mutagenesis can be used to make the deletions. Additional approaches include PCR amplification of a desired portion of the E-chitinase sequence shown in Figure 3 (SEQ ID NO. 3), exonuclease digestion techniques, chemical mutagenesis, and alanine scanning mutagenesis. Especially preferred functional fragments will include a chitinase domain as discussed above. See e.g., Ausubel et al, supra; and Sambrook et al., supra; for disclosure relating to these techniques.

In a related approach, computer-assisted methods (e.g., BLAST, FASTA, and TFASTA) can be used to identify regions of substantially amino acid homology ( greater than about 90%) between the E-chitinase amino acid sequence shown in Figure 4 (SEQ ED NO. 2) and other chitinase gene sequences and particularly bacterial or fungal chitinase gene sequences available from Genbank. As will be fully appreciated, such computer-assisted approaches can be used to identify catalytic (chitinase) domains in the E-chitinase. A particularly preferred E-chitinase fragment includes from about 100 to about 2500 to 2552 amino acids, preferably from about 500 to about 2000, more preferably about 550 to about 1000 and even more preferably about 550 to about 675 amino acids of the sequence shown in Figure 4 (SEQ ID NO. 2) as determined, e.g., by standard protein sizing techniques such as gel electrophoresis. An especially preferred fragment includes amino acids 1 to 660 of the sequence shown in Figure 4 (SEQ ID NO. 2) which fragment has been shown to bind chitin and to have significant E-chitinase activity.

As noted, minor modifications of the Vibrio furnissii E-chitinase sequence shown in Figure 3 (SEQ ID NO. 1) may result in proteins which have substantially equivalent activity as compared to the full-length E-chitinase. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as substantial chitinase activity is present. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its activity. This can lead to the development of a smaller active molecule which may have broader utility in some applications. For example, it is possible to remove amino or carboxy terminal amino acids which may not be required for the enzyme activity.

Thus, in one embodiment, the present invention includes polynucleotides with genetic alterations that do not substantially impact E-chitinase activity as determined by the standard chitinase assay (ie. less than about 5% as determined by the assay). The genetic alterations can be synthetic, i.e., can be introduced experimentally, or may be naturally-occurring, e.g., in the form of E-chitinase isoforms or strain variants.

Further provided by the present invention are polynucleotide sequences and particularly RNA sequences that are antisense. In addition, the invention also includes a polynucleotide encoding a polypeptide having an amino acid sequence as shown in Figure 4 ( SEQ ID NO.2) and having at least one epitope for an antibody immunoreactive with the enzyme polypeptide.

As noted, the present invention provides oligonucleotide primers that are complementary to the nucleotide sequence shown in SEQ ID NO: 1. In most cases, the primers will be a DNA sequence of between about 12 to about 70 nucleotides in length, e.g., about 20, 30, 40, to about 50 or about 55 nucleotides in length. The oligonucleotide primers can suitably include restriction sites to add specific restriction enzyme cleavage sites to the PCR product as needed, e.g., to introduce a ligation site. Preferred DNA oligonucleotide primers are spaced from one another in opposing direction relative to extension of the primers. That is, the primers are spaced relative to each other on a polynucleotide template (usually on different strands) sufficient to produce an amplification product of at least about 50 nucleotides, at least about 60 to about 100 nucleotides, at least about 200 to 500 nucleotides, at least about 600 to 1000 nucleotides, or at least about 1000 to about 2500 nucleotides or greater as determined, e.g., by gel electrophoresis. Synthetic methods for making oligonucleotide primers are well known in the field. Exemplary primers are provided in the examples which follow.

As noted, particularly preferred polynucleotides of this invention exhibit a length of between about 50 to about 2550 nucleotides or more, preferably between about 100, 200, 300, 400, 500, 600, 800, 1000, 1500, 2000, up to about 2550 to 2560 nucleotides as determined by standard nucleic acid sizing techniques such as agarose or polyacrylamide gel electrophoresis. The polynucleotide can be RNA (e.g., mRNA) DNA (e.g., genomic or cDNA) or a chimera thereof as desired.

As discussed, preferred polynucleotides of this invention encode an amino acid sequence that exhibits chitinolytic activity in a standard chitinase assay. That is, the amino acid sequence is capable of "functioning" in the assay to degrade (hydrolyze) chitin into component oligosaccharides. In most instances, the chitin oligosaccharides will have the formula [G1CNAC]_{N ;} in which n is usually from about 1 to about 8 and more typically from about 1 to 3. More specific small chitin oligosaccharides include N- acetyl-D-glucosamine [GlcNAc]; N,N'-diacetylchitobiose [(GlcNAc)₂ ]; tri- [(GlcNAc)₃ ], tetra- [(GlcNAc)₄] , penta- [(GlcNAc)₅ ]. hexa- [(GlcNAc)₆ ], hepta [(GlcNac)₇], and octa- [(GlcNac)₈] mers of GlcNac.

Several chitinase assays are known in the field involving colorimetric, viscosimetric, or radiometric measurments. See e.g, Boiler, T and Mauch, F. (1988) Methods in Enzymol. 161: 430; Cabib, E. (1988) Methods in Enzymol. 161: 424; Ohtakara, A. (1988) Methods in Enzymol. 161: 461-426; and references cited therein.

By the term "standard chitinase assay" or like term as used herein is meant the following radiometric assay for detecting and optionally quantitating chitinase activity. In general, the assay uses radiometry to detect formation of oligosaccharides from radiolabeled chitin. Preferred E-chitinases or functional fragments thereof of this invention will exhibit from between about 1 to about 10, more preferably from between about 2 to about 6 in the assay as nmol of ³H-(GlcNAc)₂ released from ³H-chitin per min at 37°C or nmol of p-Nitrophenol released from pNP-(GlcNAc)₂ per min at 37°C. More specific chitinase activity can be determined as nmol of ³H-(GlcNAc)₂ released from ³H-chitin per min per mg of protein at 37 °C or nmol of p-Nitrophenol released from pNP-(GlcNAc)₂ per min per mg of protein at 37°C. A preferred standard chitinase assay measures formation of water soluble oligosaccharides by the following steps: a) preparing [acetyl-³H] labeled chitin ( about 15mg/ml, specific activity about 1 X 10 ⁶ cpm/mg) in 5 micro liters of a suitable buffer (e.g., tris or phosphate buffer pH 6 to 8); b) adding an aliquot of the E-chitinase to the buffer to final reaction volume of about 100 microliters; c) incubating multiple reaction volumes at 30°C (occassional shaking is preferred) from between about 10s, 15s, 30s, 1 minute, 10 minutes, 30 minutes up to about 1 to 2 hours or more if desired; d) stopping any chitinase activity in the reaction volumes by adding about 0.2 ml of 10% trichloroacetic acid; e) centrifuging the reaction volume through Gelman glass fiber filters (2.5cm diameter is preferred), type A/E, into scintillation vials; and f) measuring any water soluble oligosaccharides in the scintillation vials by radiometery. Specific methods for preparing the ³ H-chitin, preparing and analyzing multiple reaction volumes, filtering the reaction volumes and measuring the water soluble oligosaccharides have been reported. See Cabib, E. (1988), supra, for additional disclosure relating to the standard chitinase assay.

Specific polynucleotides of this invention can be tested for capacity to encode a protein with chitinase activity in what is referred to as a "cell-mediated chitinase assay" . That assay is described in U.S. Pat. No. 5,374,540 and is useful for detecting chitinase producing bacteria. More particularly, the chitinase activity is expressed in the assay as the ratio of a culture plate clearing zone diameter in agar in minimal media supplemented with 1.0% collodial chitin as the carbon source. A value of 1.0 is taken as no clearing (ie. no chitinase) detectable in the assay. Exemplary host cells suitable for use in the assay are recombinant bacteria that express and preferably secrete an E-chitinase or a functional fragment or derivative thereof capable of producing a clearing zone in the assay. Preferred polynucleotides are capable of inducing a clearing zone of at least 20%, 30%, 40%, 50%, 60%, 70%, up to about 100% to about 200% or greater than the clearing zone produced by a suitable control strain such as E.coli or a suitable Vibrio furnissii null mutant.

Additionally preferred polynucleotides of this invention are capable of encoding an E-chitinase or functional fragment thereof with significant capacity to hydro lyze and preferably bind chitin or pNP-(GlcNAc)₂ as determined in a standard chitin binding assay. The chitin binding assay can be performed by one or a combination of different strategies employing well-known protein binding assays. For example, in one approach, chitin is detectably-labeled with a radionuchde or other detectable tag such as biotin. The detectably-labeled chitin is then allowed to contact the encoded E-chitinase or functional fragment thereof under conditions conducive to forming a binding complex. The binding complex so formed can be detected by a variety of means including centrifugation sedimentation, polyacrylamide gel electrophoresis, and radioimmunoassay, e.g., ELISA. More specific disclosure relating to preferred chitin binding assays can be found in

Morimoto K, et al. (1997) J. Bacteriol, 179(23):7306. It will be apparent to those of skill in this field that the chitin binding assay can be used to identify chitin binding domains in the E-chitinase or functional fragment thereof.

Preferred are polynucleotides that encode a polypeptide with capacity to specifically bind between about 60% to about 100% (mole amount), preferably about 70%, 80%, or 90% of the chitin bound by the E-chitinase sequence shown in Figure 4 (SEQ ID NO. 2).

By the term "specific binding" or similar term is meant a molecule disclosed herein and particularly an E-chitinase or functional fragment thereof which binds another molecule, preferably chitin, thereby forming a specific binding pair. The specific binding pair does not significantly recognize and bind to other molecules as determined by, e.g., Western blotting, ELISA, RIA, gel mobility shift assay, enzyme immunoassay, competitive assays, saturation assays or other suitable protein binding assays known in the field.

Polynucleotides of this invention are typically isolated, meaning that the polynucleotides usually constitute at least about 0.5%, preferably at least about 2%, and more preferably at least about 5% by weight of total polynucleotide present in a given fraction. A partially pure polynucleotide constitutes at least about 10%, preferably at least about 30%, and more preferably at least about 60% by weight of total nucleic acid present in a given fraction. A pure polynucleotide constitutes at least about 80%, preferably at least about 90%, and more preferably at least about 95% by weight of total polynucleotide present in a given fraction. Purity can be determined by standard methods including gel electrophoresis.

Polynucleotides of this invention can be made by a variety of conventional methods. For example, the DNA can be isolated using hybridization or computer- assisted techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or DNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features; 3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of interest; and 4) computer searches (e.g., using BLAST, FASTA or TFASTA) of sequence databases for statistically similar sequences. See e,g, Wallace, et al, Nucl Acid Res., 2:879, 1981 for a specific screening method.

In a preferred approach, polynucleotides of this invention are derived from a bacterial organism, and preferably from Vϊbrionacease and particularly Vibrio furnissii. For example, a DNA expression library can be made from Vibrio furnissii in accord with standard approaches ( e.g., lambda gtl 1 library) which library can be screened indirectly for enzyme peptides having at least one epitope, using antibodies specific for the enzyme. Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of enzyme DNA.

Isolated polynucleotides of this invention can be introduced into suitable host cells as needed. Exemplary host cells which can express the isolated polynucleotides of this invention are known in the field and include bacterial cells such as Vibrio and particularly Vibrio furnissii, andE. coli strains such as BL21(DΕ3), ER2267, MM294, DM52, XLl-Blue MR (Stratagene). Although the bacterial cells are generally preferred, in some cases it may be useful to express the polynucleotides in other cells such as animal cells (e.g., CV-1 and COS-7 cells). In addition, it is possible to express certain isolated nucleic acids of the invention in yeast cells (e.g., S. cerevisiae), amphibian cells (e.g., Xenopus oocyte), and insect cells (e.g., Spodoptera frugiperda and Trichoplusia ni). Methods for expressing isolated and recombinant DNA in these cells are known. See e.g., Sambrook et al., Molecular Cloning (2d ed. 1989), Ausubel et al. supra, and Summer and Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures: Texas Agricultural Experimental Station Bulletin No. 1555, College Station Texas (1988). Specifically preferred bacterial host cells are discussed more fully below.

In embodiments where bacterial expression of a polynucleotide is desired, specific expression vectors may be advantageously selected depending upon the use intended for the expressed protein. For example, when large quantities of the enzyme are to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Those which are engineered to contain a cleavage site to aid in recovering are preferred. Such vectors include but are not limited to the E. coli expression vector pUR278 (Ruther et al, EMBO J. 2:1791,

1983), in which the enzyme coding sequence may be ligated into the vector in frame with the lac Z coding region so that a hybrid -lac Z protein is produced; pIN vectors (Inouye &

Inouye, 1985, Nucleic acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.

Chem. 264:5503-5509); and the like. The examples below provide preferred host cells and vectors of the invention.

Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired. The polynucleotides of this invention can be readily made by techniques well known in the field including those techniques involving large-scale production thereof such as those including use of roller bottles, bioreactors and the like. Preferred are techniques suitable for commercial scale production of the polynucleotides or polypeptides encoded by same.

The term "complementary" or like term refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified.

Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 95% of the nucleotides of the other strand, usually at least about 98%, and more preferably from about 99 to about 100%. Complementary polynucleotide sequences can be identified by a variety of approaches including use of well-known computer algorithms and software.

It is preferred that the polypeptides of the present invention be substantially pure. That is, the polypeptides have been isolated from cell substituents that naturally accompany it so that the polypeptides are present preferably in at least 80% or 90% to 95% homogeneity (w/w). Polypeptides having at least 98 to 99% homogeneity (w/w) are most preferred for many pharmaceutical, clinical and research applications. Once substantially purified the polypeptide should be substantially free of contaminants for therapeutic applications. Once purified partially or to substantial purity, the polypeptides can be used therapeutically, or in performing a desired assay. Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis. Particularly preferred polynucleotides and polypeptides of this invention are provided as substantially or totally sterile formulations.

The term "substantially homologous" is also used herein with reference to relationship between two polypeptide sequences and generally refers to subunit sequence similarity between the two molecules. When a subunit position in both of the polypeptides is occupied by the same monomeric subunit, i.e. an amino acid sometimes referred to as an amino acid residue, then they are homologous at that position. Homology between the two sequences is a direct function of the number of matching or homologous positions, e.g., if 50% of the subunit positions in the two polypeptides are homologous then the two sequences are 50% homologous. By "substantially homologous" is meant largely but not wholly homologous. More particularly, the term is meant to denote at least about 70% or greater homology as defined above with respect to the V furnissii E-chitinase sequence illustrated in SEQ ID NO: 2. Preferred polypeptides of the invention have between about 75% to about 99% or greater homology, preferably about 80%, 85%, 90%, 95%, 97%, 98% or greater homology as defined above with respect to the V. furnissii E-chitinase sequence illustrated in Figure 4 (SEQ ID NO: 2).

The E-chitinase polypeptides of the present invention can be separated and purified by appropriate combination of known techniques. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel- filtration, and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electrical charge such as ion-exchange column chromatography, methods utilizing specific affinity such as affinity chromatograph, methods utilizing a difference in hydrophobicity such as reverse-phase high performance liquid chromatograph and methods utilizing a difference in isoelectric point, such as isoelectric focusing electrophoresis, metal affinity columns such as Ni-NTA. See generally Sambrook et al. and Ausubel et al. supra for disclosure relating to these methods. Additionally, the full-length E-chitinase shown in Figure 4 (SEQ ID NO. 2) or functional fragment thereof can be covalently linked (ie. fused) to specific amino acid sequences to enhance isolation or solubility. Illustrative of those amino acid sequences include what are often referred to as "derivatives" such as polyhistidine (6XHIS), MYC, EE, ect.

Preferred polypeptides of this invention generally exhibit a molecular weight from about 60, 65, 70, 75, 80, 85, 90, 95 to about 100 kDa as determined by standard protein sizing manipulations such as polyacrylamide gel electrophoresis or centrifugation sedimentation. Additionally preferred are those polypeptides that are substantially homologous to the E-chitinase sequence shown in Figure 4 (SEQ ID NO: 2) as determined by the methods described earlier. Particularly preferred are those polypeptides having from between about 100 to about 3000 amino acids, preferably about 600 to about 2550 amino acids as determined by conventional protein sizing and sequencing manipulations.

Additionally preferred is a substantially pure enzyme preparation including at least the isolated E-chitinase of this invention. The substantially pure enzyme preparation may also include at least one other hydrolyzing enzyme such as those specifically referenced herein. Also typically included are additives such as buffers, salts, enzyme stabilizers (e.g., serum albumin) and the like to promote activity of the enzyme preparation. Preferred are substantially pure enzyme preparations comprising the E-chitinase sequence shown in Figure 4 (SEQ ID NO. 2) although for some applications it may be useful to employ functional fragments or derivatives of the sequence show in Figure 4 (SEQ ID NO. 2).

In one embodiment, the substantially pure enzyme preparation includes the Vibrio furnissii E-chitinase. In this embodiment, the substantially pure enzyme preparation can be used to degrade chitin into component chitin oligosaccharides. In preferred embodiments, the E-chitinase is provided in the preparation from about 0.01%, 0.02%, 0.05%, 1%, 2%, to about 5% by weight. The substantially pure enzyme preparation may be provided as a lyophilized powder or as a liquid as described.

In another embodiment, the substantially pure enzyme preparation includes the Vibrio furnissii E-chitinase and at least one other chitin catabolizing enzyme such as the periplasmic chitodextrinase (Endo-I), the periplasmic β-GlcNAcidase (Exo-I), the cytoplasmic β-GlcNAcidase, or the aryl β-N-acetylglucosaminidase (Exo-II) from Vibrio furnissii or functional fragments thereof. The enzymes have been disclosed, e.g., in the published PCT Application No. WO 96/25424. See also Keyanhi, N.O and Roseman, S., (1996) J. Biol. Chem, 271 33414; and Keyanhi, N.O and Roseman, S., (1996) J. Biol. Chem., 271 33425. The amount of each enzyme in the preparation will depend on intended use but will generally be provided from about 0.01%, 0.02%, 0.05%, 1%, 2%, to about 5% by weight.

A substantially purified enzyme preparation of this invention includes essentially any of the proteins and particularly E-chitinases or functional fragments thereof of this invention prepared from recombinant clones or isolated from a suitable host organism.

The term "amino acid sequence" as used herein generally refers to any polymer preferably consisting essentially of any of the 20 naturally occurring amino acids regardless of its size. Although the term "protein" is often used in reference to relatively large proteins, and "polypeptide" or "peptide" is often used in reference to small amino acid sequences, use of these terms in this field often overlaps. Thus, it will be understood that the term generally refers to proteins, polypeptides, and peptides unless otherwise noted.

As discussed, the present invention features novel methods for producing a substantially pure preparation of a desired chitin oligosaccharide and particularly N, N'- diacetylchitobiose [(GlcNAc)₂ ]. Preferred use of the methods include use of any of the E-chitinases, functional fragments or derivatives disclosed herein. For example, a desired E-chitinase can be provided in several forms as needed. In one embodiment, the E- chitinase can be provided as a substantially pure enzymatic preparation as described above. In another embodiment, the E-chitinase can be provided by a recombinant cell and particularly a bacterial cell such as E. coli expressing the E-chitinase. In embodiments where the E-chitinase is provided by recombinant cells, it is generally preferred that the cell secrete the enzyme. However, in instances where a particular recombinant cell does not secrete the enzyme at suitable levels, a cell lysate can be prepared from the cells to enhance chitinolytic activity. See Example 2 below for a preferred procedure for making a cell lysate that includes the E-chitinase.

As noted, the present methods for making chitin oligosaccharides can employ nearly any of the E-chitinases disclosed herein as well as functional fragments thereof. For example, in one embodiment, the full-length E-chitinase shown in Figure 4 (SEQ ED NO. 2) can be used to make the oligosaccharides. In another embodiment, the methods can be used with an amino acid sequence that is substantially homologous to the E- chitinase shown in Figure 4 (SEQ ID NO. 2). The substantially homologous enzyme may include iso forms, ie. naturally-occurring variants of the sequence shown in Figure 4 (SEQ ID NO. 2). Particularly, the substantially homologous E-chitinase may include amino acid deletions (contiguous or noncontiguous), additions, or substations with respect to the E-chitinase sequence shown in Figure 4. In a related embodiment, a functional fragment of the E-chitinase shown in Figure 4 (SEQ ID NO. 2) can be employed. Preferred E-chitinases (or functional fragments) for use in the methods will exhibit at least about 50%, 60%, 70%, 80%, 90%, up to about 100% of the activity of the E-chitinase illustrated in Figure 4 (SEQ ID NO. 2) as determined by the standard chitinase assay.

Choice of a particular E-chitinase or functional fragment thereof for use in the present methods will be guided by intended use. For example, some bacterial strains may express and secrete a particular E-chitinase fragment more efficiently than another under the same culture conditions. In general, the following steps can be used to facilitate production of a substantially pure chitin oligosaccharide or a desired mixture of chitin oligosaccharides: (1) conversion of chitin to a mixture of soluble oligosaccharides, (GlcNAc)_n in which n is 1, 2, 3, 4, 5, 6, 7, 8 or greater, preferably between about 1 to 3, and (2) resolution of the oligosaccharides in a substantially pure preparation. The following Table I below summarizes preferred methods for making specific chitin oligosaccharides. The methods are flexible and can be readily adapted isolate a particular chitin oligosaccharide or a mixture thereof:

TABLE I GENERAL METHODS FOR PREPARING CHITIN OLIGOSACCHARIDES¹

^!See also the U.S. Patent No. 5,792,647; the pending U.S. Application No. 08/600,452; and the published PCT Application No. WO 96/25424. The pending U.S. Applications and the published PCT application disclose an E-chitinase encoded by the chiA gene from Aeromonas hydrophila. The E-chitinase of the present invention is encoded by the chiE gene from Vibrio furnissii.

² The normal substrate for egg white lysozyme is the N-acetylmuramyl glycosidic bond in bacterial cell walls, but it cleaves (GlcNAc)₆ at about 50% of this rate. The rates of cleavage of other (GlcNAc)_n (relative to (GlcNAc)₆) are as follows: (GlcNAc)₆, 100; (GlcNAc)₅, 13; (GlcNAc)₄, 2.6; (GlcNAc)₃, 0.33; (GlcNAc)₂, 0.001. Preferred use of the present methods employs as a starting material a purified or semi-purified preparation of chitin. As noted, chitin is a naturally-occurring polymer composed of β- 1,4 linked polysaccharides of GlcNac. Chitin is available in substantial amounts, e.g., from shells of various Crustacea, from the shells of certain insects, and from cell walls of specific fungi, diatoms, and other microorganisms. In a more preferred embodiment, a desired amount of crude commercial chitin is acidified to provide a colloidal preparation of semi-purified chitin. See Pegg, G.F. (1988) Methods in Enzym. 181 (Part B) 484.

In one embodiment, the semi-purified colloidal chitin is combined in a suitable reaction mixture with an E-chitinase (or functional fragment) of this invention. The amount of the chitin and the E-chitinase to be used will be guided by several parameters including the amount and type of soluble chitin oligosaccharide desired. In a preferred embodiment, from between about 10 grams to about 100 grams or more of the semi- purified chitin is combined with from between about 2 grams to about 25 grams (wet weight) of a crude lysate made from a bacterial cell culture that has been transformed with an E-chitinase of this invention. In a particularly preferred embodiment, the E- chitinase is the amino acid sequence disclosed in Figure 4 (SEQ ID NO. 2). Preferred methods for making the bacterial lysate are described in more detail below. Reaction conditions can be adapted as needed, but will generally be sufficient to produce the chitin oligosaccharide mixture desired.

In a more preferred embodiment of the method, a mixture of soluble chitin oligosaccharides having the formula: [(GlcNAc)_n ] in which n is 1 or 2 with trace quantities of trisaccharide, is obtained by exhaustive chitinolysis. By exhaustive chitinolysis is meant incubation with the E-chitinase from about 6 hours, 12 hours, lday, 2days, to about 3 days or more using a reaction temperature from between about 30° C to about 40°C, and typically about 37°C. Preferred buffer conditions include but are not limited to use of a volatile buffer such as pyridine-acetate, pH6.5. The chitinolytic reactions described above in which the E-chitinase or a functional fragment or derivative thereof is used can be monitored by one or a combination of conventional strategies, e.g, to fine tune reaction conditions or to identify production of particular chitin oligosaccharides of interest. For example, the chitin oligosaccharides in a given reaction mixture can be readily identified and quantified, if desired, using standard chromatographic approaches such as paper or thin layer chromatography, gel filtration, gel filtration HPLC (high performance liquid chromatography), reverse-phase HPLC, and salt precipitation. Purity may also be monitored by GC-MS (gas chromatography-mass spectrometer interface) according to standard methods if needed. Pure chitin oligosaccharides suitable for use as controls can be obtained from several sources such as Hae Seikaguku Co. More specific disclosure relating to methods for detecting chitin oligosaccharides can be found, e.g., in U.S. Patent Nos. 5,705,634; 4,804,750; Bassler, B. L. et al. supra, and references cited therein.

In a specifically preferred embodiment, substantially all of the GlcNAc and the

GlcNAc trisaccharide is removed from the mixture of soluble chitin oligosaccharides described above. In this embodiment, the end product desired is a substantially pure preparation of [(GlcNAc)₂ ]. By the term "substantially all" is meant removal of from about 90%, 95%, up to about 99% or greater of the chitin mono- and trisaccharide as determined, e.g., by HPLC. The undesired mono- and trisaccharide can be removed by one or a combination of approaches. In one embodiment, the mixture of chitin oligosaccharides is subjected to a purification step that is sufficient to remove substantially all of the unwanted chitin oligosaccharides.

In one embodiment of the purification step, the removal of the chitin mono- and trisaccharide is achieved by chromatography such as gel filtration, HPLC, or a combination thereof. Briefly, the reaction mixture described earlier is subjected to HPLC or gel filtration and HPLC under chromatographic conditions conducive to separating the chitin disaccharide from the mono- and trisaccharides. In another embodiment of the purification step, the chitin trisaccharide is specifically removed in accord with methods described in the U.S. Patent No. 5,792,647; the pending U.S. Application No. 08/600,452 and the published PCT Application No. WO 96/25424. For example, in one embodiment of the disclosed methods, recombinant periplasmic β-N-acetylglucosaminidase (Exol) can be used to remove the trisaccharide. The Exo I enzyme can be provided as a cell lysate or as a substantially pure enzyme as desired. Preferably, substantially all of the chitin trisaccharide is removed by the Exo I enzyme as determined, e.g, by HPLC.

In a preferred embodiment of the purification step, the reaction mixture is subjected to conditions that facilitate rapid isolation of the chitin disaccharide while minimizing use of chromatography and related procedures. In a specifically preferred approach, the reaction mixture comprising the chitin oligosaccharides is combined with specific recombinant E. coli cells that are capable of catabolizing the chitin monosaccharide in the reaction mixture but are not fully capable and preferably are unable to catabolize the chitin disaccharide. Most wild-type E. coli strains can utilize the chitin disaccharide as a carbon source. Genetic methods were used to isolate mutant E. coli strains that are unable to use the chitin disaccharide but were newly able to utilize chitin monosaccharide (GlcNAc). A particularly preferred approach for isolating such E. coli mutants has been disclosed. See Example 7 below.

The present invention is more fully understood by comparing the fermentative characteristics of E. coli and V. furnissii. For example, mutant E. coli strains can only utilize GlcNAc, whereas wild type V. furnissii can utilize (GlcNAc)_n, where n = 1-4 without using special methods for induction. Higher oligomers such as (GlcNAc)₅,

(GlcNAc)₆ and chitin are also consumed by V. furnissii, but only after special conditions of induction. Intact induced V. furnissii cells consume 0.32 μmole GlcNAc/mg protein/min at 25°C, which is about the same as the maximum rate of glucose utilization by E. coli at 37°C. (GlcNAc) and (GlcNAc)₃ are consumed at about the same rate (per GlcNAc equivalent) by V. furnissii. (GlcNAc)₄ is catabolized more slowly. (GlcNAc)₅ and (GlcNAc)₆ are not utilized unless the cells are selectively induced on swarm plates. Accordingly, at least certain Vibrio cells are able to ferment specific chitin oligsaccharides in accord with the present methods. See Bassler, B.L., et al. (1991) J. Biol. Chem. 266: 24276 and Bassler, B.L., et al. (1991) J. Biol. Chem. 266: 24268.

In a more specifically preferred embodiment of the purification step, the recombinant E. coli strain Xm.1.4 is combined with the mixture of chitin oligosaccharides under conditions suitable for fermenting essentially the monosaccharide. The Xml.4 strain is described below in Example a. In particular, suitable conditions include growth of X_ml .4 in LB or minimal salts plus a suitable amount of GlcNAc to mid-log phase followed by harvesting and washing the cells in minimal media and adding the harvested cells to an incubation mixture comprising the oligosaccharides at 37°C. Incubation time will vary depending on several parameters including the amount of recombinant E. coli cells added and the amount of chitin monosaccharide to be fermented. However, usually about 4 to 6 hours will be sufficient. The resulting mixture will be referred to herein as a semi-purified (GlcNac)₂ preparation.

In accord with the present methods, a substantially pure preparation of the chitin disaccharide can be achieved by one or a combination of different approaches. In one embodiment, the chitin disaccharide is purified by subjecting the mixture of chitin oligosaccharides to chromatography such as discussed above, e.g, gel filtration, HPLC, or a combination of gel filtration and HPLC.

However for most applications, it is preferred to purify the chitin disaccharide by precipitation. In one embodiment, the semi-purified (GlcNac)₂ preparation described above is deproteinized, combined with an ion exchange resin, preferably a mixed bed ion exchange resin, and concentrated to a syrup from which the chitin disaccharide can be obtained by crystallization by combining the chitin disaccharide mixture with a lower alcohol or a mixture of lower alcohols. The deproteinization can be achieved by a number of standard procedures including extraction with a base and an acid, or by mixing with suitable organic solvents. In some cases, it may be helpful to treat the mixture of chitin oligosaccharides to loosen and degrade the proteins. Preferred ion exchange resins Dowex AG1 and Dowex 50W. The recrystallization step can be conducted 1, 2, 3, or more times to enhance purity if desired although a single recrystallization step will suffice for most applications.

As used herein, the term lower alcohol means a branched or straight chain alcohol consisting of between 1 to 5 carbons (inclusive), e.g., ethanol, propanol, iso-propanol, butanol, iso-butanol, tert-butanol, pentanol, iso-pentanol, and the like with methanol being generally preferred.

As noted, the present invention can be adapted to obtain a substantially pure preparation of a desired chitin oligosaccharide of a mixture thereof. See Table I above. In embodiments where preparation of a substantially pure chitin disaccharide is of interest, the yield thereof will vary depending on several parameters such as the quantity and quality of the semi-purified chitin used as the starting material and the purity of the chitin disaccardide desired. However, in most instances, practice of the present methods can provide an amount of substantially pure chitin disaccharide equal to about 10% to about 50% and more typically about 25% of the wet weight of the semi-purified chitin used as the starting material.

It will be apparent from the foregoing that the methods of the present invention are readily adjustable for industrial scale production of a desired chitin oligosaccharide or mixture of chitin oligosaccharides. Specific methods for the industrial scale handling of chitin and chitin oligosaccharides are known in the field and include the commercial scale preparation of chitin. See e.g., U.S. Patent No. 5,705,634 for disclosure relating to commercial preparation of chitin. Related methods suitable for practicing the present invention on a large scale include use of industrial scale reaction vessels such as bioreactors to degrade large quantities of chitin with the E-chitinases or functional fragments thereof disclosed herein. Such methods can be employed in accord with the present invention to produce industrial scale quantities of substantially pure [(GlcNAc)₂ ] or other chitin oligosaccharides if desired.

As previously discussed, the present invention also relates to methods for enhancing genetic manipulation of specific prokaryotes and more particularly to methods for introducing foreign DNA into V. furnissii. In accordance with the present invention, it was noted that it was difficult to genetically complement certain V. furnissii mutants using genetic material propagated in E. coli. It appeared that the problems were exacerbated by introduction of foreign DNA into V. furnissii. For example, where efficient transformation of foreign DNA was desired, e.g., by electroporation, very low transformation frequencies were often obtained. According to the invention, it was discovered that the Vibrio had an R-M system which damaged or destroyed foreign DNA introduced into the cells.

The following describes general steps for overcoming the V. furnissii R-M system:

1. Isolation of a V. furnissii DNA restriction enzyme: A V. furnissii DNA restriction enzyme was isolated according to the standard methods. Briefly, specific

V. furnissii cell fractions were tested for restriction enzyme activity. Using standard DNA restriction enzyme testing procedures, a restriction enzyme was isolated. A preferred

DNA restriction enzyme testing procedure involved digesting a bacterial plasmid (e.g,. pBR322) with a desired restriction enzyme, e.g., Sau96I, separating any DNA segments on an agarose or polyacrylamide gel, and then determining the identity of any restriction cleavage sites.

2. Cloning of a V. furnissii methylase modification gene: The second step was to clone a V. furnissii DNA methylase modification gene that is capable of protecting unmethylated (e.g., E. coli) DNA against the restriction enzyme identified in step 1. The methylase was cloned along lines disclosed by Kiss, A. et al. (1985) Nuc. Acids. Res. 6402. In general, the method involves expression of a DNA methylase gene and upon transformation, any plasmid within a DNA library which contains and expresses the cloned methylase gene will methylate its cognate recognition site(s). When the library is subsequently selected by cleavage with a restriction endonuclease of an appropriate specificity, the modified plasmids should not be restricted (cleaved) and remain viable upon a second transformation step. Other plasmids not including the methylase gene should be restricted by the restriction endonuclease and therefore transform at a much reduced efficiency. See also U.S. Pat. Nos. 5,434,068; 5,534,428 for more specific disclosure relating to the method of Kiss et al., supra.

3. Transformation with the V.furnissii methylase gene. A recombinant transformation vector encoding the V.furnissi methylase gene isolated in step 2 above was introduced into E. coli strains that were used to propagate the V. furnissii DNA banks. Foreign DNA propagated in the transformed E. coli strains was found to be highly resistant to degradation by the V. furnissii R-M system as determined by standard DNA restriction enzyme testing.

It will be apparent from the foregoing and the examples that follow that the present methods for genetically manipulating V.furnissii can be readily adapted to introduce foreign DNA into nearly any prokaryotic cell comprising at least one R-M system and preferably one or two R-M systems. The methods, as adapted, generally include identifying in the prokaryote the DNA restriction enzyme(s) that are sensitive to DNA methylation. See step 1 above and also McClelland, M et al. (1994) Nucl Acids. Res. 22: 3640 for DNA restriction enzymes that are known to be sensitive to DNA methylation. The method of Kiss et al., supra, described in step 2 above can be used to identify the corresponding DNA methylase gene. See also Genbank (National Library of Medicine, 38 A, 8N05, Rockville Pike, Bethesda, MD 20894). Genbank is also available on the internet at http://www.ncbi.nlm.nih.gov.) for a list of prokaryotic DNA methylases. Also in accord with step 2 above, a DNA fragment that includes the DNA methylase gene can be readily obtained. Transformation of E. coli with the DNA methylase gene according to step 3 above, can provide a recombinant E. coli strain that is useful for propagating desired gene banks or other nucleic acids. Nucleic acid that has been propagated in the E. coli strain (now modified by methylation or hemi-methylation) can be isolated and checked for resistance to the DNA restriction enzyme(s) expressed by the prokaryotic cells by conducting a standard DNA restriction enzyme assay. Modified nucleic acid showing resistance to the DNA restriction enzyme(s) can be introduced into the prokaryotic cells by any desired route including transformation or transconjugation. Successful introduction of the modified nucleic acid will be manifested, e.g., by transformation efficiency ratios in the range of from about 10⁵ to about 10⁶ transformants /μg modified DNA; and transconjugation efficiencies in the range of from about 0.5 to about 0.8 transformants per recipient cell.

The present invention is further illustrated by the following Examples. These examples are illustrative of the present invention and should not be construed as a limitation thereof.

EXAMPLE 1- Isolation and characterization of the V.furnissii E-chitinase encoded by the chiE gene.

A. Isolation of the V. furnissii E-chitinase

The laboratory strain V.furnissii 7225, or "wild type", was originally shown to express a number of β-hexosaminidases. One of these, Endo I, is a chitodextrinase (Keyhani,N.,et al. (1996) J. Biol. Chem. 271: 33414). While this enzyme is not a chitinase, it has over-lapping specificities with E-chitinase, e.g., they both hydrolyze the synthetic substrate MUF-(GlcNAc)₂ . A "wild type" strain, however, designated SRI 519, was found to express the E-chitinase, but not Endo I. Southern-blotting hybridization with P DNA probes for endl showed that this gene is deleted in SRI 519. A genomic library was therefore prepared from SRI 519 since clones that tested positive with MUF- (GlcNAc)₂ would indicate expression of chiE and not endl. A cosmid library was constructed by using bacterial genomic DNA from V.furnissii SRI 519. Library construction, including conditions for partial genomic DNA digestion (using Sau 3 A) and ligation into the cosmid vector SuperCosl(A"&aI, CEP and Bam HI) were performed according to the supplier's recommendation (Stratagene). The ligation mixture was packaged into phage by using GigaPack Gold III extract (Stratagene) and transfected into E.coli XLl-Blue MR strain according to the supplier's recommendations. The screening procedure was carried out with MUF-(GlcNAc)₂ and MUF-GlcNAc as described (Keyhani,N.,et al. (1996), supra.. 10 independent clones were MUF-(GlcNAc)₂ positive and MUF-GlcNAc negative, the desired phenotype. Restriction analysis of these cosmid clones showed that all of them were derived from the same region of t eV.furnissii chromosome and contained inserts of approximately 30-40 kb. Sublconing of Hind III and Pst I fragments into the pUC19 vector (Fig. 2) revealed that the gene encoding MUF-(GlcNAc)₂ positive activity was located at the 3' flank of a 14 kb Hind III fragment and in the middle of a 6.5 kb Rst I fragment.

Resfriction analysis and functional mapping of the gene led us to construct a plasmid designated pENDOII, which contained the smallest Sad-Hind III fragment (2.5 kb) encoding an ORF with MUF-(GlcNAc)₂ positive activity. Sequence analysis of this 2.5 kb fragment revealed an ORF encoding a 69 kDal protein, but also that this protein is truncated at its C-terminus. BLAST sequence analysis showed that the ORF has a high level of homology to class 18 hexosaminidases including a number of known bacterial and viral chitinases. Comparison restriction analysis of the 6.5kb Pst I and 2.5 kb Sac I- Hind III fragments showed that largest ORF in the 6.5kb Pst I fragment contained 1.8 kb more DNA, i.e., that the ORF in the Sac l-Hind III fragment was truncated at its 3' end. Since both ORF expressed activity with MUF-(GlcNAc)₂, the truncated Sac l-Hind III ORF was designated endoll, while the full length gene was called chiE.

Colloidal chitin plate assays showed that the clone containing pENDOII (Sac I- Hind III, 2.5kb) does not clear chitin, but the clone containing pChiE (Pst I, 6.5 kb) does. Sequence analysis of the 3 '-terminal 1.8 kb showed that this fragment encoded the C- terminal domain, or the presumptive chitin-binding domain of Chi E. The complete ORF for chiE encodes an 89 kD protein. Upstream of chiE is an ORF similar to tryptophanase, followed by a stop codon. There are 200 bp of a putative regulatory region between the stop codon and the beginning of chiE.

B. Characterization of the V. furnissii E-chitinase

The isolated E-chitinase gene was sequenced by the double sfranded primer walking method described above and was found to comprise a sequence of 2553 base pairs. The entire nucleotide sequence is shown in Figure 3 (SEQ ID NO:l). The V. furnissii DNA fragment contained one major open reading frame. The predicted amino acid sequence of the E-chitinase encoded by the chiE gene is shown in SEQ ID NO:2 and consists of 851 amino acids with a predicted molecular weight of 89.848kDa.

The predicted amino acid sequence is similar to other bacterial chitinases, which are in Class 18 of the glyocosyl hydrolases (Henrissat, B and Bairoch, A. (1993)

Biochem. J. 293, 781). Our data, indicate that V.furnissii E-chitinase contains an N- terminal signal sequence, a catalytic domain and a chitin binding domain.

EXAMPLE 2- Purification of recombinant E-chitinase from V.furnissii

The isolated V. furnissii E-chitinase described in Example 1 can be purified by a variety of procedures. A preferred procedure is as follows:

Step 1: Crude Extract- 1.5 liters each of LB medium, supplemented with 100 μg/ml ampicillin in two 6-liters flasks, were inoculated with 100 ml of overnight culture of E. coli BL21 harboring the plasmid pChiE7. The culture was shaken vigorously at 37°C until A ₆₀₀ was about 3.0, and the cells harvested by centrifugation at 4000 X g for 10 min at 4°C. The following steps were conducting at 0-4°C unless otherwise stated.

The cell pellet was washed twice with 800 ml of M9 media, and cells were resuspended in Buffer A (lOmM Tris-HCl, 50 mM NaCI ImM DTT, ImM EDTA, 5mM MgCl_2; 1% PMSF, 20mM ,- amino-caproic acid ( pH 7.4)) and disrupted by three passages through a French Press.

Step 2: Streptomycin Sulfate Precipitation-Nucleic Acids were precipitated with a solution of streptomycin sulfate (160 μl of 10% stock/ml crude extract), which was added dropwise with stirring. The white precipitate was removed by centrifugation at 235,000 X g for 60 min and discarded.

Step 3: Affinity adsorption of chitinase to chitin- 10 ml of 10% colloidal chitin in buffer A was added to 40 ml of crude extract, and incubated on a rotor for 60 min to adsorb chitinase E. The colloidal chitin was washed three times with buffer B (lOmM Tris-HCl, 800 mM NaCI ImM DTT, ImM EDTA, 5mM MgCl_2> 1% PMSF, 20mM ,- amino-caproic acid ( pH7.4)) to remove unbound proteins (centrifugation). The chitin pellet was resuspended in 20 ml of buffer A and transfered to a dialysis bag. Digestion of the chitin and dialysis against 4 L of Buffer A was carried out at room temperature for 24 h with three changes of the buffer. The solution contained soluble ChiE.

Step 4: FPLC Ion -Exchange Column Chromatography-The apparently homogeneous preparation was concentrated by centriprep and applied to a 5-ml bed volume monoQ column for purification by FPLC. A 50 -800 mM NaCI gradient

(lml/min) was used in a solution containing 10 mM Tris-HCl, ImM DTT, ImM EDTA, 5 mM MgCl , 1% PMSF, 20mM ,- amino-caproic acid ( pH7.4). The active fractions were eluted between 300 - 400 mM of NaCI.

Throughout the fractions samples were assayed both for activity and by SDS-

PAGE. The final preparations were stable for at least several months at -80°C.

The purified E-chitinase described above can be subjected to SDS-PAGE gel electrophoresis to verify the molecular weight of the recombinant protein. Additional molecular weight determinations can be performed by gel filtration if desired. Preferred enzymatic conditions for using the isolated E-chitinase are as follows: a pH of about 6.0 to 7.0, a reaction temperature of between about 37-42°C, and a reaction buffer including about 50-100 mM NaCI.

EXAMPLE 3- Preparation of [(GlcNAc)₂ ] using the V furnissii E-chitinase The following method was used to make the chitin disaccharide, (GlcNAc)₂, from chitin. Crude commercial chitin (1 kilogram) was dissolved in concentrated HC1 at 37°C, filtered and reprecipitated by dilution in ice water. This step removes many impurities, and gives a finely divided, almost colloidal preparation of the chitin.

The V. furnissii E-chitinase described in Examples 1 and 2 was used to produce

[(GlcNAc)₂ ] by the following method. The method was particularly useful for making large scale amounts (ie. gram amounts) of the chitin oligosaccharide. About 100 g of partially purified chitin was digested with a crude extract of an E. coli transformant containing the cloned E-chitinase. See Example 1 for methods of making the E. coli transformant. The major product was (GlcNAc)₂ contaminated with significant quantities of GlcNAc and smaller amounts of (GlcNAc)₃. The latter could be converted to the mono- and disaccharide with the cloned periplasmic β-N-acetylglucosaminidase (Exo I) described above, in the U.S. Patent No. 5,792,647; the pending U.S. Application No. 08/600,452; and the published PCT Application No. WO 96/25424. However, this step was found to be unnecessary because (GlcNAc)₃ is removed during crystallization of the disaccharide. The major problem was to remove GlcNAc. This problem was readily solved by employing an an E. coli mutant unable to ferment the disaccharide. See Example 7 below. The culture was deproteinized using (H⁺ form of Dowex 50WX8, treated with mixed bed ion exchange resin Biorad Dowex AG1X8), and concentrated to a syrup, from which (GlcNAc)₂ was crystallized by adding 300 ml of MeOH. After one recrystallization, about 25 g of pure disaccharide were obtained as determined by TLC, GC-MS, and HPLC.

Crude E-chitinase was prepared as follows: Cells were grown to stationary phase, harvested and washed, and stored at -70°C until used. When desired, cells were thawed, ruptured by passage through a French Press, centrifuged for 10 min at 10,000 x g to remove cell debris. Nucleic acids were removed from the supernatants with streptomycin

(see below), and the chitinase precipitated with 70% saturated ammonium sulfate. The crude preparation was dissolved in the reaction buffer (50 mM pyridine acetate, pH 6.5).

Additional methods for preparing chitin oligosaccharides have been described, e.g., in the PCT application WO 96/25424 and are as follows:

1. Partial acid hydrolysis of particulate chitin yields a mixture of soluble oligomers, some of which are partially deacetylated. The mixture is then quantitatively reacetylated with acetic anhydride in water (Roseman, S. and Ludowieg, J. (1954) J. Am. Chem. Soc. 76: 301. and Roseman, S. and Daffner, I. (1956) Anal. Chem. 28: 1743).

2. A mixture of lower oligosaccharides, (GlcNAc)-,, n = 2-4, and possibly some (GlcNAc)₅ can be produced by the action of lysozyme on chitin. Egg white lysozyme is plentiful, commercially available, and quite inexpensive (about $10/gram).

3. Table I above presents related examples of procedures that can be used to prepare chitin oligosaccharides. These methods will provide large quantities of pure oligosaccharides. Suitable recombinant enzymes and/or intact cells can be used to resolve oligosaccharide mixtures.

EXAMPLE 4- Characterization of a resfriction-modification (R-M) system in V. furnissii.

A. Demonstration of an R-M system in V.furnissii Electroporation of V. furnissii in the presence of plasmids such as pACYCl 84, propagated in E. coli HB101, yielded 0-20 transformants/μg DNA, compared with 5xl0⁵ transformants/μg DNA when plasmids were grown in V. furnissii. It was discovered that V. furnissii has a restriction-modification (R-M) barrier against foreign DNA, and that the organism is sensitive to electroporation. B. Isolation of a V. furnissii DNA restriction enzyme:

The restriction enzyme was isolated as follows: 10 g of cell paste were ruptured in a French Press, treated with streptomycin to remove nucleic acids, and the enzyme isolated by two successive chromatographic steps, a phosphocellulose column followed by an hydroxyapatite column. Using standard restriction enzyme testing procedures, the V.furnissii enzyme was found to be an isoschisomer of Sau96I (commercially available from New England Biolabs, Beverly, MA). A preferred DNA restriction enzyme testing procedure involved digesting a bacterial plasmid (e.g., pBR322) with a desired restriction enzyme, e.g., Sau96I, separating any DNA segments on an agarose or polyacrylamide gel, and then determining the identity of any restriction cleavage sites

The shortcomings of the R-M system have been reduced or eliminated by steps described in examples which follow.

EXAMPLE 5- Isolation of a V.furnissii methylase gene.

The V. furnissii methylase gene (MVfuI) was cloned and expressed in E. coli. A methylase selection method was employed which method has been used to clone a number of restriction-modification (R-M) systems (Kiss, Posfai, et al. 1985 ID: 3122). The procedure can select such clones even when they occur at frequencies as low as 10 ^"3 to 10^"4 within a complex library.

In this example, V.furnissii DNA was partially digested with Sau 3A1, and ligated into the Bam HI site of the vector pBR325 (see Fig. 6). The plasmids were used to transform E. coli ER2267 (New England Biolabs). From 3-4000 independent clones were isolated and challenged with Sau 961. Undigested plasmids were used for a second transformation, and 20 of the resulting clones were analyzed. Five of these contained the same 2.75 Kb insert, and were partially resistant to Sau 961, suggesting that they contained the desired methylase gene. Since only partial protection of the plasmid DNA was obtained against Sau 961, expression of the enzyme was increased by cloning MVfuI (Nhe l/Sal I fragment) into the high copy number vectors pUCl 9 and pUCl 8 at the Sal llXba I site, giving pMVful 8 and pMVful9 (pUC18 and pUC19 are also used to determine the orientation of cloned genes). pMVful9 and pMVfulδ showed different levels of methylation. pMVfulδ DNA was completely protected against Sau 961 digestion because the gene is under control of the plac promoter, whereas the methylase gene in pMVful9 was in the opposite orientation, and the lowered quantity of methylase led to only partial protection of the DNA. With these results, the desired plasmid, pMVful29 was constructed from pMNful as shown in Fig. 6.

An important property of the two component system just described is that pMVful29 in Fig.6 contains the origin of replication ori 101. This ori is compatible with most other commonly used plasmid origins of replication, such as P15, colEl, and ori V. Therefore the E. coli host used for plasmid propagation can be doubly transformed, one plasmid carrying the methylase gene (pMVful29) and the other carrying the gene to be protected by methylation prior to introducing it into V. furnissii or other organisms that express similar R-M systems.

EXAMPLE 6 - Transformation and transconjugation in V. furnissii

The effectiveness of the two component system described in Example 5 was assayed by measuring the efficiency of transformation and of transconjugation of V. furnissii with vectors propagated in the doubly transformed E. coli cells. The results are shown in Table II below. At least three independent measurements were obtained in each experiment.

A. Transformation

Doubly transformed E. coli HB 101 was used for propagation of plasmids for testing with V.furnissii cells. The plasmids used to transform the E. coli cells were: (1) pMVful29 (methylase +) or pWSK129 (methylase -); (2)the test vectors pACYC184 (contains ori pl5, which is compatible in bothE. coli, and V.furnissii) or pBR322 (contains ori col ΕI, which is compatible in E. coli but incompatible in V.furnissii). Double transformants were selected by antibiotic resistance, Km and Tc. After overnight growth in the presence of IPTG to induce the plac promoter, plasmid DNA was purified and elecfroporated into freshly prepared V. furnissii cells. Transformants were incubated for at least 1 hr at 37°C in LMB media, and then plated on LMB agar plates containing 50 μg/ml ampicillin and 15 μg/ml tetracycline. Ampicillin is lethal to E. coli , while Tc is lethal to all V. furnissii cells except those containing methylated test plasmids carrying the Tc^r gene.

Table II below shows that pACYC184 was protected by the two component system. The efficiency of transformation was increased by 4-5 orders of magninide, sufficient for doing genetic experiments in V.furnissii.

B. Transconjugation

For transconjugation, test vectors were used such as pSF4, which contained the necessary origins of replication, oriT for transconjugation, and pi 5 for replication, compatible with V.furnissii. E. coli SI 7-1 was used as the host because it expresses the mobilization factors required for transconjugation to organisms such as V. furnissii. The approach was similar to that described above. E. coli SI 7-1 was transformed with pSF4 and with either ρMVful29 (methylase +) or ρWSK129 (methylase -).

The doubly transformed E. coli cells are prepared as described in (a) above (Km^rTc^r), mixed with an equal number of wild type V. furnissii cells, and plated overnight at 30°C on LMB agar broth to permit transconjugation. The mixture of cells was harvested, diluted, and plated on LMB agar containing 50 μg/ml ampicillin and 15 μg/ml tetracycline. Survivors showing this phenotype should be primarily transconjugants containing appropriately methylated DNA. The efficiency of transconjugation can be extraordinarily high, and is measured as the number of survivors per V.furnissii cell used in the experiment. The value 1.0 denotes 100% efficiency. The Table shows, in fact, that given a compatible origin of replication, 80% efficiency was obtained by the two component protection system.

Table II is shown below:

Table II Efficiency of transformation and transconjugation of V.furnissii

EXAMPLE 7 - Production of V.furnissii chiE null mutants

The most promising "suicide" vector approach for constructing recombinant site- directed mutants is the method of Simon, et al. (Simon, R.U. et.al. (1983) Biotechnology 1 :784). This procedure involves conjugal transfer of plasmids from E. coli S17-1 containing a mobilizing donor (IncP-type) to any Gram negative bacterium. In our case the bacterium was Vibrio furnissii SRI 519. A vector, pNQ705 was constructed from pBR322 in which its origin of replication was deleted and replaced with R6K ori: pNQ705 also carries Cm^r and a multiple cloning site. pNQ705 can only be replicated in cells containing π, a protein encoded by the pir gene. An E.coli 1 pir lysogen is used to amplify the plasmid, which is then mobilized into recipient cells where the plasmid cannot replicated, but can be incorporated into the genome by recombination of the gene carried by the plasmid and the corresponding gene in the genome. Antibiotic resistant cells are therefore recombinants, and are null mutants, since the gene in the genome has been interrupted with an antibiotic cartridge.

Vectors used in the method are shown in Fig. 5A-5B. First, the suicide vector was modified by insertion of the SacB gene from pLOl to have a positive selection marker for second round of recombination . Second, the 0.6 kb Nru l-Sma I fragment from pENDOII (see above) was inserted into the Eco RN site between Cm ^r and the SacB genes. The final plasmid pΝQ-ΔChiE-SacB was propagated in E.coli SI 7-1 and transconjugated to V.furnissii SRI 519. 7 independent recombinant Ap ^r Cm ^r clones were isolated . All showed a Chi E negative phenotype by the colloidal chitin -plate assay. Southern-blotting hybridization with several [³²P]-DΝA probes derived from pNQ-ChiE- ΔSacB confirmed that all of the inserts are in the same region of the chromosome (chiE region), and each insert comprises the entire pNQ-ΔChiE-SacB sequence.

A. Complementation of the chiE phenotype in null mutant A V.furnissii chiE clone, AF100, was isolated as described above, and was used in the following experiment. Another null mutant, CY101, that contained a site-specific deletion of exol gene served as a control. In this mutant, the ChiE function has been lost, suggesting a link between the regulatory regions in the two genes. The 6.5 kb Pst fragment from pChiE was cloned into the pSF4 plasmid ( Selvaraj, G., Fong,Y.C, Iyer, V.N. (1984) Gene 32:235), resulting in pSF/CHIE (Fig 5B ). This plasmid contains the complete chiE ORF under control of its native promotor. Also it has Tc ^r drug selection marker and mobilization site for transconjugation. pSF/CHIE was transformed into E.coli AFIOIM for in vivo modification and succsessfully transconjugated into AF100 and CY101. As a negative control we used the pSF4 plasmid. The resulting strains AF102, AF103 and AF104 (see Table III) showed different behavior on colloidal chitin plates. The pSF4 vector could not restore the chitin clearing phenotype in either mutant. On the other hand, the cloned chiE restored the chitin clearing phenotype in the chiE null mutant, but not in the control, the exol null mutant. This was additional evidence that AF100 has a specific mutation in the chiE gene. The regulatory system also functioned normally in the cloned chiE transconjugant since it was subject to glucose catabolite repression, as is the wild type.

Table III is shown below:

TablelH [ please provide citations under "Source" in Tables III and IV]

The following Table IV describes various vectors including those specifically referenced herein.

Table IV

References for Tables III and IN:

1. Studier, F.W. et al. - (1990) Meth.Enzym. 185:60.

2. Raleigh, E.A. et al. (1996/97) ΝEB catalog.

3. Milton, D.L. et al., (1992) J. Bacteriology, 174:7235. 4. Cowell, R.R. (1984) Vibrios in the Environment, John Wiley and Sons, Inc., New York.

5. Bolivar F. et al. (1977) Gene. 2(2):95.

6. Prentki P. et al. (1981) Gene. 14(4):289.

7. Yanisch-Peπon C. et al. (1985) Gene. 33(1): 103. 8. Selvaraj G. et al. (1984) Gene. 32(l-2):235.

9. Keen NT. et al. (1988) Gene. 70(1):191.

10. SuperCosl. Instruction Manual. (1997) Stratagene Inc.

11. Wang RF. Kushner SR. (1991) Gene. 100:195.

12. Lenz O. et al. (1994) J. Bacteriology. 176(14):4385.

As disclosed in the published PCT Application No. WO 96/25424, the so-called

"suicide vector": approach was also employed to construct the suicide vectors, pNQT:EndoI::Cm and pNQT:Exol::Cm. The constructs contained the following: (a) Ori R6K, an origin of replication that requires the protein for replication; (b) the Mob RP4 genes that permit the plasmid to be transferred (mobilized) into any Gram negative recipient such as V. furnissii; (c) a Tc^r, or tetracycline resistance gene and (d) the fragment of DNA encoding endol or exol interrupted with the Cm or chloramphenicol resistance gene.

Two strains of V.furnissii were used as recipients of the conjugations, V.furnissii

SR1519 (wild type) and V.furnissii AP801, a mutant in nagE (the GlcNAc permease) that has been described (Bastec. et al. supra). A similar protocol was followed for constructing pNQT:Exol::Cm and the corresponding null mutants. The deletion mutants were characterized by Southern blots, which showed that the Cm^r cartridge had been inserted in the proper position in the V.furnissii genomic DNA.

EXAMPLE 8- Catabolism of chitin disaccharide N,N'-diacetylchitobiose in E. coli and V. furnissii. It was unexpectedly found that wild-type E. coli strains actively grows on (GlcNAc) , and that the catabolic pathway is encoded by the so-called cryptic eel (cellobiose) operon. The results were discussed in Keyhani, N.O. and Roseman, S. (1997) RN4S (USA) 94: 14367 the disclosure of which is incorporated herein by reference.

Transposon mutants of E. coli were isolated that were unable to ferment the disaccharide, but grew normally on GlcNAc. The mutants were used to screen an E. coli genomic cosmid library for restoration of (GlcΝAc)₂ fermentation. A partial sequence of the complementary DNA fragment mapped the clone to the (previously sequenced) E. coli genome between 39.0-39.2'. The ORF in this region had previously been assigned to code for a "cryptic" cellobiose (eel) utilization operon.).

Three E. coli strains were used to analyze use of the disaccharide:

1. XLl-Blue MR: this strain grows on (GlcNAc)₂ as the sole source of carbon, and swarms (chemotaxis) to the disaccharide.

2. Xml .4- this strain was isolated by transposon mutagenesis of the wild type with a mini-mμ lac-tetracycline transposable element. The mutant could not grow on, ferment or swarm to (GlcNAc) , but utilized GlcNAc as efficiently as the parent strain.

3. Xml .4:pΕS 1 - subcloning of a cosmid library led to the isolation of a 7.3 kb DNA fragment from E. coli that complemented Xml.4 in each phenotype.

Induced and uninduced cells were tested for β-GlcNAcidase activity with PNP-

GlcNAc. Hexosaminidase activity was observed only with induced wild type (XLl-Blue MR), and with Xml .4:pES 1.

The following materials and methods were used, as needed, in the Examples 1-8 above. 1. Buffers

The composition and pH (at room temperature, unless otherwise noted) of several buffers used herein are listed in Table V below:

Table V

Buffer Composition

EP (electroporation buffer) 10% glycerol

Transformation buffer 50 mM CaCl₂, 10 mM Tris-Cl, pH 7.5

TE 10 mM Tris-Cl, 1 mM EDTA, pH 8.0 TAE 40 mM Tris-acetate, 1 mM EDTA, pH 8.0, diluted from 50X stock

Improved TBE

127 mM Tris, 235 mM boric acid, 2.52 mM

EDTA, pH 8.3, dilute from 10X stock

SSC 0.064 M NaCI, 0.012 M Na citrate, pH 7.5

SHM Used for "stringent" hybridization. 25 mM Na phosphate, pH 7.5, 5X SSC, 5% instant Carnation milk, 40% deionized formamide, 0.1 mg/ml sonicated salmon sperm DNA.

2. Bacterial Culture Media Reagents used to prepare bacterial media were purchased from Difco Labs (Detroit, MI). The formulations of the culture media used in this study are listed below in Table VI. TABLE VI

Medium Composition (g/1) Artificial Sea Water NaCI, 23.6; Na₂SO₄, 4; NaHCO₃, 0.2; KC1, (ASW)0.66; KBr, 0.96; H₃BO₃, 0.026; MgCl₂.6H₂O, 10.6; SrCl₂.6H₂O, 0.04; CaCl₂, 1.48; K₂HPO₄, 0.04; NH4CI, 2.0;

Hepes-50% ASW Hepes buffer, 11.9 (50 mM) pH

7.5; in 50% ASW

Lactate-ASW D,L-lactate, 5; in Hepes-50% ASW LB Bacto-tryptone, 10; yeast extract, 5; NaCI, 10

LMB Bacto-tryptone, 10; yeast extract, 5; NaCI, 20

Marine Medium 2212 Bacto-peptone, 5; yeast extract, 1.0; in Hepes-50% ASW

MacConkey

Bacto-Peptone, 17; Proteose Peptone, Agar 3; Bile Salts, 1.5; NaCI, 5; Neutral Red, 0.075; Crystal Violet, 0.5; Bacto Agar, 15

M9 Na₂HPO , 6; KH₂PO , 3; NaCI, 0.5; NH4CI, 1; MgSO , 0.24; CaCl₂, 0.015; carbon source, 2; Thiamine-HCl, 0.002; casamino acids, 2

Medium A KH₂PO₄, 4.5; K₂HPO₄, 10.5; (NH- 2SO4, 1; MgSO₄, 0.12; carbon source, 2; Thiamine-HCl, 0.002

Antibiotics were used in the following concentrations: ampicillin, 15 μg/ml (30 μg/ml for agar plates) and tetracycline, 5 μg/ml (10 μg/ml for agar plates).

3. Bacterial Strains

V. furnissii 7225 (available from the ATCC), a wild type strain which is also designated V.furnissii SRI 514, was maintained at room temperature in a soft agar slab consisting of (g/1): yeast extract, 3; bactopeptone, 10; NaCI, 10; and agar, 5, in Hepes- buffered 50% ASW (see below). E. coli strains K-12, HB101, BL21(DΕ3) and XL-Blue were stored as frozen cultures in LB. Typically, sfrains were grown overnight in rich broth (plus appropriate antibiotics for cells containing plasmids) with vigorous shaking. Fresh medium was inoculated with cells from the overnight culture at a 1 :20 or 1 :50 dilution, and this culπire was grown to the desired density, usually mid-exponential (OD₅₉₀ = 0.3-0.4).

4. Preparation of DNA

Genomic DNA was prepared from V.furnissii SRI 519 by standard procedures. See e.g., Ausubel et al., supra.

5. Plasmid Purification

Plasmids were prepared by the alkaline lysis method (Blaak, H., et al. supra). Cells harboring the plasmid of interest were grown in LB or M9 medium containing the appropriate antibiotic. The cells were then harvested by centrifugation, and were resuspended in buffer (150 mM NaCI, 10 mM Tris-Cl pH 8) at 15 ml buffer per g wet weight of cells. The cells were lysed at room temperaπire by the addition of 2/3 volume of 40 mM EDTA pH 8 with 1% SDS and 1 mg/ml pronase, and the cell debris was removed by centrifugation at 150,000 x g. The nucleic acids were precipitated from the supernatant fluid by the addition of 1/3 volume 40% PEG 3350 in 2M LiCl, 20 mM Tris- Cl pH 8, 2 mM EDTA. This nucleic acid pellet was homogenized in 2.5 M LiCl, 10 mM Tris-Cl pH 8, 2 mM EDTA and cooled to -20°C to precipitate RNA, which was removed by centrifugation at 250,000 x g. Finally, plasmid DNA was precipitated from the supernate with 2.5 volumes of cold EtOH. The plasmid pellet was washed with 70% EtOH to remove residual salts and was dissolved in TE buffer. The nucleic acid concentration (and relative level of protein contamination) was determined by measuring the A₂₈₀ and A₂₆₀ of the preparation, where 1.0 A₂₆₀ = 50 μg DNA. For large scale plasmid preparations, cells were grown in 1 liter of medium, while for minipreps, cells were grown overnight in 10 ml of medium. Typically, 300-700 μg of plasmid was obtained using the large-scale protocol, and 5-10 μg from the miniprep protocol. The alkaline lysis method is as follows: The cells are lysed in alkaline SDS, which denatures genomic and plasmid DNA. After neutralizing, the plasmid DNA is selectively renatured, and purified by treating with RNAase A, phenol/chloroform, chloroform/isoamyl alcohol, and precipitated with ethanol or PEG.

6. Bacterial Transformation

The heat shock procedure described in Maniatis et al. ((Adam, R., et al., supra) was used. Host cells were grown to mid-exponential phase using an overnight culture started from a single colony. Plasmid DNA (5-50 ng in TE buffer) or DNA from a ligation mixture (10-100 ng in ligation buffer suggested by ligase manufacnirer) was added (1-2 μl) to a cell suspension of 50-100 μl on ice. Occasionally, the DNA in ligation mixtures was precipitated by adding 1/10 volume of 3 M sodium-acetate, pH 4.6, and 2 volumes of ice cold ethanol, followed by incubation of the samples at -70°C for 20 min. The resultant pellet was washed once with an equal volume of 70% ethanol, dried and resuspended to 10-20 1 TE prior to use in transformation reactions. Cells with the DNA were heat shocked for 1 min at 42°C or for 3-5 min at 37°C in sterile glass tubes, 0.5-1.0 ml of LB was immediately added to the tubes and the cells were allowed to recover for 30-60 min at 37°C with vigorous shaking. The transformed cells were then plated on selective media. Transformation efficiency was usually monitored by using a known amount of a control plasmid (pBR322).

An alternate transformation procedure involving electroporation was also used. The Cell-Porator system from GIBCO-BRL and the manufacturer's recommended procedures were used (See, e.g., Sambrook et al., supra). The Cell-Porator consists of a system for placing a suspension of cells and plasmids between two electrodes. Brief unidirectional electrical pulses render the cell membranes temporarily permeable to the DNA. Mid-exponential cells grown in LB were harvested and washed with EP and resuspended to 1/100 volume of the original culture in EP. These cells were either used immediately or frozen for later use. DNA (10-50 ng in 1-2 μl) was added to 30 μl of cells. The electroporation settings used were those recommended by the manufacturer supra). Efficiency was determined as described in the heat shock procedure.

7. Restriction Enzyme Digestions and Analysis of DNA Standard procedures were followed (Ausubel et al, supra; and Sambrook et al., supra) for restriction enzyme digestions and analysis of the fragments generated by these digestions. Generally, 0.5-1 μg of DNA, purified as described, was digested with 1-5 U of the desired restriction enzyme under the conditions suggested by the manufacturer. In situations where digestion by more than one enzyme was desired, the digests were usually performed separately; the DNA was precipitated (by the addition of 1/10 volume 2.5 M NaOAc and 2.5 volumes of cold EtOH), dried, and the second digest was then performed. When double digestions were performed, the first enzyme used was the one requiring a lower concentration of salt; in this manner, inhibition of the second restriction enzyme (by salts remaining from the first digest) was minimized. The resulting DNA fragments, in BPB/Ficoll tracking dye, were separated by electrophoresis through 0.8% agarose gels in TAE buffer. Agarose gels were 13.4 x 14.2 x 0.5 cm submerged horizontal gels. The gels were run at 4-5 V per cm until the BPB dye was 2-3 cm from the bottom of the gel. DNA within the gel was visualized by soaking the gel in a 0.1 μg/ml solution of ethidium bromide for 20 min, followed by rinsing in H₂O for 10 min. The gel was photographed under UV illumination with a Polaroid Land Camera (Polaroid Type 667 film). A Hindlll digest of 8 DNA was used for molecular weight standards.

DNA fragments were eluted from Agarose gels using standard techniques including electroelution, purification using QIAEX II (Qiagen, Inc., Chatsworth, CA), and the band intercept method GeneCleanll comprises a silica matrix to which DNA in cell extracts is adsorbed under conditions of high ionic strength. The matrix is washed free of protein and other contaminants, and highly purified DNA is eluted at increased temperature, low ionic strength. Ligations were performed using standard conditions. Blunt-end ligations were performed at 18°C for 18 hr, whereas compatible overhanging ends were incubated with ligase for 2 hr at 25°C. Inserts in cloning experiments were purified from gels as described above and ligated to phosphatase-treated vector that had also been cut to produce compatible ends in a ratio of 2-5 : 1.

pBR322 was often used as the vector herein as was pUC18, pUC19, and pVex. pVex is a high copy number plasmid with a T7 polymerase promoter near its multiple cloning site, thus allowing for overexpression of the desired gene product. The polymerase is generated in the host cell E. coli BL21(DΕ3) by induction with EPTG.

Thus, in experiments involving ligations of cloned DNA fragments into pVex, induction of expression by IPTG indicates that the cloned gene is in proper orientation with respect to the T7 polymerase promoter.

8. DNA Sequence Analysis

The DNA prepared from the recombinant clones was sequenced by the dideoxy method using a U.S. Biochemical Sequenase® sequencing kit (Blaak, H., et al. supra and Blaiseau, P.L. and Lafay, J.F. (1992) Gene. 120: 243). The kit provides buffers, labeling mixtures, termination dideoxy nucleoside triphosphates, and T7 DNA polymerase. Plasmid preparations were used in double-stranded sequencing according to the manufacturer's recommended procedures.

9. Molecular Analysis and Sequencing of DNA

Preparation and analysis of DNA, Southern bio ting, filter colony hybritization, restriction enzyme digestion, ligation and transformation were performed using standard techniques (See e.g., Ausubel, et al. supra ).

The DNA prepared from the recombinant clones was sequenced by dideoxy terminators method using a USB Sequenase^R sequencing kit manually as well as automatically on Applied Biosystem model 373 A at the JHU Sequencing facility, with appropriate primers and series of subclones. The nucleotide and protein sequence data was assembled and analysed with the GCG sequence analysis package Version7, Genetics Computer Group, Madison, Wi. Homology search in GenBank and Swiss Protein were carried out with BLAST and FASTA programs.

10. DNA Hybridizations

DNA fragments were hybridized to one other, by the method of Southern (Blaak, H., et al. supra), to ascertain whether they contained the same or different genes. The DNA fragments were cut from the respective plasmids with restriction enzymes and gel purified as described above. The samples were heated at 65°C for 10 min, and 6 ng each loaded per lane of a 1% Agarose gel. Following electrophoresis, the gel was washed sequentially with 0.1 M HC1 (10 min), 0.5 M NaOH + 1.5 M NaCI (2 x 15 min), and 0.5 M Tris, pH 7.4 + 1.5 M NaCI (2 x 15 min). A Southern transfer to nitrocellulose was performed overnight in 0.64 M NaCI, 0.12 M Na citrate, pH 7.5. The blot was allowed to dry and the original gel stained with ethidium bromide to determine whether all of the DNA had been transfeπed. The blots were then probed as follows. Labeled probes were prepared from the cloned genes by the random primer method (Blaak, H., et al. supra), using a BMB Random Priming Kit® (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and α-[ P]-dCTP. The kit contains standard DNA, hexanucleotide mixture containing all possible sequence combinations of hexanucleo tides, deoxynucleoside triphosphates, and Klenow enzyme. One or more of the random hexanucleotides hybridize with the fragment to be labeled, and a strand complementary to the DNA is synthesized with labeled nucleotides (not provided in the kit) by extension of the hexanucleotide with the Klenow fragment of DNA polymerase I. After purification

(TCA precipitation, Sephadex columns), the specific activities of the probes were 10⁸-10⁹ cpm per μg DNA. The probes (at least 10⁶ cpm aliquots each) were denatured, and hybridized to the membranes. Hybridization conditions varied from stringent to reduced stringency as follows (only the extremes are given): 65°C overnight in 6X SSC buffer, 0.5% SDS, 5X Denhardt's solution (30), and 100 μg calf thymus DNA per ml; 37°C, 6X SSC, 10% dextran sulfate, 35% formamide. The blots were washed three times (10 min each) in 5X SSC, 0.1% SDS at 25°C, then for 60 min in 0.5 SSC, 0.1% SDS at 40°C. The blots were finally exposed to X-ray film.

11. Chitinase Assays

Enzyme activity was qualitatively determed from clones growing on agar plates by use of synthetic analogue MUF-(GlcNAc)₂ which, when hydro lyzed, yields a fluorescent product, 4-methylumbelliferone. Colonies were lifted on to sterile Whatman No.l paper, which was then sprayed with 0.6 mM MUF-(GlcNAc)₂ in 10 mM sodium phosphate buffer, pH 7.5. The paper was incubated at 37°C for 10-20 min and sprayed with saturated sodium bicarbonate solution in order to enhance the fluorescence yield. Colonies expressing the chiE gene product glowed bright blue when illuminated under a UV source.

12. Discontinuous and Continuous Spectrophometric Assays

Performed as described previously (Keyhani,N.,et al.-(1996) J. Biol Chem. 271,33414.

13. Colloidal chitin plate assay

Colloidal chitin was used in an overlay of agar growth plates. 1 ml of a 10% suspention of colloidal chitin was spread on top of 10 ml agar media. Inocula were either streaked or spread on the surface of agar. Enzymatic activity was detected as clear zones around the colonies or streaks.

14. Solubilization of [³H]-Acetyl-Chitin Radioactive acetylation of chitosan was performed by a modification of the method of Cabib (Cabib 1988: Methods Enzymology 161: 424). Completely deacetylated chitosan was dissolved in water and precipitated by dropwise addition of saturated NaHCO solution. The precipitated chitosan was washed three times in water before final dissolution in 10% acetic acid. The pH of the chitosan/acetic mixture was titrated to 6.0 with saturated NaHCO₃ before addition of methanol to 10% (v/v) final concentration. The sample was then placed on ice and acetic anhydride in acetone (5 mCi ³H-acetic anhydride) was added with stirring; the sample was then kept on ice for 8 hours with stirring, at which point an additional 10-fold molar excess of unlabeled acetic anhydride (as 1 : 1 mix with acetone) was added, and the mixture kept overnight at 0-4°C. During the reaction, chitin precipitated out of solution. The labeled chitin was exhaustively dialyzed against 10% HO Ac, 1% HO Ac and finally against deonized water. The chitosan amino group N-acetylation exceeded 95%, and the specific activity of the labeled product was 2- 5xl0⁴ cpm/μg.

The standard chitinase assay mixture contained 50 mM phosphate buffer, pH 7.5,

50 mM NaCI, 0.1 mg/ml BSA, and 60-80 μg of ³H-Chitin (1-5x10° cpm) in a total volume of 0.5 ml. Assay tubes were preincubated at 37°C prior to addition of the enzyme, incubated for lOmin to lh at 37°C, and the reaction terminated by heating at 100°C for 5 min. Denaturated proteins and chitin were pelleted by centrifugation at 12,500 X g for 5 min in a Beckman microfuge; 350 μl aliquots of the supematants were added to 2 ml

Ultra Gold XR liquid scintillation cocktail and counted for 1-5 min in a Packard 2200CA Tricarb Liquid Scintillation analyzer.

15. Product characterization The products of hydrolysis were assayed by modifying the published procedures for separating the oligosaccharides on TLC and HPLC (Bassler et.al. (1991) J.Biol. Chem. 266: 24268). GlcNAc and chitin oligomers ranging from GlcNAc to (GlcNAc)₆ can be separated and quantified using an ISCO model 2350 HPLC system with a TosoHaas Amido 80 column (25cm X 4.6mm) and a Hewlett Packard 3390A Integrator. A guard column with Thomson TSK guard gel Amide 80 cartrige was also used. The solvent system consisted of 70% acetonitrile: water, using a flow rate of 0.8 ml/min with no washing of the column between injections. Oligosaccharides were detected at A₂₀₅ using an ISCO V ⁴ Variable Wavelenght Absorbance Detector. Retention time for the single, sharp peaks (min) were: GlcNAc=6.65, (GlcAc)₂=7.78, (GlcNAc)₃=9.09, (GlcNAc)₄=10.73, (GlcNAc)₅=12.61 and (GlcNAc)₆=14.99. The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modification and improvements within the spirit and scope of the invention as set forth in the following claims.

All references described herein including research publications and issued U.S. patents are incorporated herein by reference.

Claims

What is claimed is:

1. An isolated polynucleotide encoding an extracellular chitinase (E- chitinase) wherein the polynucleotide is substantially homologous to the nucleic acid sequence of Figure 3 (SEQ ID NO. 1) or the complement thereof.

2. The polynucleotide of claim 1 , wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 1.

3. The polynucleotide of claim 1, wherein the polynucleotide encodes a polypeptide substantially homologous to the sequence of Figure 4 (SEQ ID NO: 2).

4. The polynucleotide of claim 1 , wherein the extracellular chitinase (E- chitinase) hydrolyzes chitin or a chitin oligusaccharide in a standard chitinase assay.

5. The polynucleotide of claim 4, wherein the standard chitinase assay detects N-acetyl-D-glucosamine (GlcNAc), and β, l-> 4-linked oligomers thereof.

6. The polynucleotide of claim 5, wherein the β, l-» 4-linked oligomers include at least N N'-diacetylchitobiose [(GlcΝAc)₂ ].

7. The polynucleotide of claim 6, wherein the extracellular chitinase (E- chitinase) has a molecular weight of between about 60 to about 100 kDa as determined by polyacrylamide gel electrophoresis using sodium dodecyl sulfate (SDS).

8. The polynucleotide of claim 1 , wherein the polynucleotide is a DNA or RNA.

9. An isolated polynucleotide that hybridizes to the sequence of SEQ ID NO: 1 or the complement thereof under moderate stringency conditions.

10. The polynucleotide of claim 9, wherein the nucleic acid hybridizes to the sequence of SEQ ID NO: 1 or the complement thereof under high stringency conditions.

11. A recombinant vector comprising the polynucleotide of claim 1.

12. The recombinant vector of claim 11, wherein the vector is pSF/ChiE.

13. The recombinant vector of claim 11 , wherein the vector is pChiE.

14. A host cell comprising the recombinant vector of claim 11.

15. The host cell of claim 14, wherein the host cell is an E. coli strain BL21.

16. A isolated extracellular chitinase (E-chitinase) from Vibrio furnissii capable of hydrolyzing chitin in a standard chitinase assay.

17. A substantially pure enzyme preparation comprising at least the isolated E-chitinase of claim 16.

18. The isolated E-chitinase of claim 16, wherein the standard chitinase assay detects N-acetyl-D-glucosamine (GlcNAc), and β, l-» 4-linked oligomers thereof.

19. The isolated E -chitinase of claim 18, wherein the β, 1— > 4-linked oligomers include at least N, N'-diacetylchitobiose [(GlcΝAc)₂ ].

20. The isolated E-chitinase of claim 19, wherein the E-chitinase has an apparent molecular weight of between about 60 to about 100 kDa as determined by polyacrylamide gel electrophoresis using sodium dodecyl sulfate (SDS).

21. The isolated E-chitinase of claim 20, wherein the E-chitinase has at least about 70% amino acid sequence identity to the sequence shown in Figure 4 (SEQ ID NO: 2).

22. The isolated E-chitinase of claim 21, wherein the E-chitinase is identical to the amino acid sequence of SEQ ID NO: 2.

23. A Vibrio furnissii null mutant lacking extracellular chitinase (E-chitinase) activity as determined by a standard chitinase assay.

24. A Vibrio furnassii null mutant lacking extracelluler chitinase (E-chitinase) as determined by a cell-mediated chitinase assay.

25. Vibrio furnissii strain AF 100.

26. A method of isolating an extracellular chitinase (E-chitinase) from Vibrio furnissii, the method comprising culturing the host cell of claim 14 in medium under conditions capable of expressing the E-chitinase in the host cell or the medium and isolating the E-chitinase from the host cell or medium.

27. A method of producing a substantially pure preparation of N N'- diacetylchitobiose [(GlcΝAc)₂ ], the method comprising the steps of: a) contacting chitin with a Vibrio furnissii extracellular chitinase (E-chitinase) under conditions sufficient to form an oligosaccharide preparation comprising N- acetyl-D-glucosamine (GlcNAc), N, N'-diacetylchitobiose [(GlcΝAc)₂ ], and a

GlcNAc trisaccharide [(GlcNAc)₃ ], b) removing substantially all of the N-acetyl-D-glucosamine (GlcNAc) and the GlcNAc trisaccharide [(GlcNAc)₃ ] from the preparation; and c) producing the substantially pure preparation of the N, N'-diacetylchitobiose [(GlcΝAc)₂ ] from the preparation.

28. The method of claim 27, wherein, in step a), the Vibrio furnissii extracellular chitinase (E-chitinase) is provided by a prokaryotic cell transformed with a recombinant vector comprising sequence encoding the chitinase.

29. The method of claim 28, wherein the prokaryotic cell is E. coli.

30. The method of claim 27, wherein in step b), the N-acetyl-D-glucosamine (GlcNAc) is substantially removed from the oligosaccharide preparation by exposing the preparation to prokaryotic cells capable of consuming the N-acetyl-D-glucosamine (GlcNAc).

31. The method of claim 30, wherein the prokaryotic cells comprise an E. coli strain capable of selectively utilizing the N-acetyl-D-glucosamine (GlcNAc).

32. The method of claim 31 , wherein the method does not substantially utilize a disaccharide.

33. The method of claim 31, wherein the E. coli strain is Xm.1.4.

34. The method of claim 27, wherein step a) further comprises deproteinizing the oligosaccharide preparation.

35. The method of claim 34, wherein step a) further comprises contacting the oligosaccharide preparation with an ion exchange resin.

36. The method of claim 35, wherein step b) further comprises crystallizing the N, N'-diacetylchitobiose [(GlcΝAc) ] from the oligosaccharide preparation by adding a lower alcohol thereto.

37. The method of claim 36, wherein the lower alcohol is methanol.

38. The method of claim 27, wherein in step a), the Vibrio furnissii E- chitinase comprises a substantially pure preparation of Vibrio furnissii extracellular chitinase (E-chitinase).

39. The method of claim 38, wherein step b) further comprises contacting the oligosaccharide preparation formed in step a) with Vibrio furnissii periplasmic β-N- acetylglucosaminidase (Exo I) under conditions capable of hydrolyzing the GlcNAc trisaccharide [(GlcNAc) ].

40. The method of claim 39, wherein step b) further comprises exposing the oligosaccharide preparation to prokaryotic cells comprising an E. coli strain capable of selectively utiliziing the N-acetyl-D-glucosamine (GlcNAc).

41. The method of claim 40, wherein the E. coli strain is Xm.1.4.

42. A method of transforming prokaryotic cells with a recombinant vector in which the prokaryotic cells comprise a restriction-modification system, the method comprising:

a) introducing the recombinant vector into first prokaryotic cells comprising a recombinant sequence comprising a prokaryotic DNA methylase gene, b) maintaining the first prokaryotic cells under conditions sufficient to methylate the recombinant vector in the cells, c) isolating the methylated recombinant vector from the first prokaryotic cells; and d) introducing the methylated recombinant vector into second prokaryotic cells under conditions sufficient to transform the second prokaryotic cells with the recombinant vector.

43. A method of transconjugating a recombinant vector into prokaryotic recipient cells comprising a restriction-modification system, the method comprising: a) introducing the recombinant vector into prokaryotic donor cells comprising a recombinant sequence comprising a prokaryotic DNA methylase gene, b) maintaining the prokaryotic donor cells under conditions sufficient to methylate the recombinant vector in the cells, c) contacting the prokaryotic donor cells with the prokaryotic recipient cells under conditions conducive to transconjugation; and d) transconjugating the recombinant vector from the prokaryotic donor cells to the prokaryotic recipient cells.

44. The method of claim 42 or 43, wherein the recombinant sequence comprises a Vibrio furnissii DNA methylase gene.

45. The method of claim 42, wherein prior to step a), the recombinant sequence is isolated by DNA methylase selection and the first prokaryotic cells are transformed with the recombinant sequence under conditions capable of expressing the DNA methylase gene in the first prokaryotic cells.

46. The method of claim 41 , wherein prior to step a) the recombinant sequence is isolated by DNA methylase selection and the prokaryotic donor cells are transformed with the sequence under conditions capable of expressing the DNA methylase gene in the prokaryotic donor cells.

47. The method of claim 42, wherein the methylated recombinant vector of step c) is resistant to cleavage by restriction endonuclease Sau96l.

48. The method of claim 43, wherein the first and second prokaryotic cells include E. coli and Vibro furnissii, respectively.

49. The method of claim 40, wherein the prokaryotic donor and recipient cells include E. coli and Vibro furnissii, respectively.

50. A recombinant E. coli strain comprising a sequence comprising a Vibrio furnissii DNA methylase gene.

51. E. coli strain AFIOIM.

52. E. coli strain AF102.