WO2003045156A1

WO2003045156A1 - Goat pregastric esterase and its use in the production of cheese

Info

Publication number: WO2003045156A1
Application number: PCT/US2001/044516
Authority: WO
Inventors: Paul L. Bolen; Paul L. Cihak; Lewis G. Scharpf, Jr.
Original assignee: International Flavors & Fragrances Inc.
Priority date: 2001-11-28
Filing date: 2001-11-28
Publication date: 2003-06-05
Also published as: AU2002217917A1

Abstract

Compositions and methods for production of enzyme modified cheeses are provided. The compositions are isolated kid goat pregastric esterases, derivatives of the isolated kid goat pregastric esterase, or a recombinant form of the kid goat pregastric esterase. These compositions are shown to produce fatty acids from fats in ratios similar to those produced using kid goat rennet preparations.

Description

GOAT PREGASTRIC ESTERASE AND ITS USE IN THE PRODUCTION OF CHEESE

Background of the Invention

Esterases, also referred to as lipases, are enzymes that cleave triglycerides or esters into carboxylic acids, or fatty acids, and mono- and di-glycerides . A pregastric esterase is an esterolytic or lipolytic enzyme secreted by the oral tissues of mammals. Animal esterases in an unpurified form called rennet have been used in the production of dairy food products and, in particular, the production of enzyme-modified cheeses or EMCs (Fox, P.F. and J. Law. 1991. Food Biotechnol . 5:239-262; Richardson, G.H. et al . 1970. J. Dairy Sci . 54:643- 647; U.S. Patent No. 2,531,329). For example, cheeses such as Romano and Provolone have a "peppery" or "piccante" flavor due to the fatty acid composition created by the enzyme in the rennet paste (Nelson, J.H. et al. 1976. J^". Dairy Sci . 60:327- 362) . Traditionally, EMCs are prepared by esterases in a rennet paste or powder obtained from the gullet of slaughtered animals. The rennet is used to treat whey to impart flavor into the cheese product.

Kid goat pregastric esterase (kPGE) in rennet paste is contaminated with other proteins found in the gullet of the goat, as well as other substances used in the preparation of the rennet. Uncontaminated or purified recombinant kPGE is useful in the production of EMCs which are acceptable to kosher and vegetarian consumers, or those who desire animal- free products .

Summary of the Invention

An object of the present invention is to provide an isolated kid goat pregastric esterase comprising SEQ ID NO:

2 which is free of other kid goat proteins. The kid goat pregastric esterase can be produced by purifying kid goat gullet tissues or by recombinant genetic expression of the protein in a non-kid goat cell, wherein the cell can be a bacterial cell, a fungal cell, a yeast cell, or an animal cell. Another object of the present invention is to provide an isolated polypeptide comprising a derivative amino acid sequence of the isolated kid goat pregastric esterase of SEQ ID NO: 2. By "derivative" for purposes of the present invention, it is meant a polypeptide which differs in amino acid sequence as compared to SEQ ID NO: 2 or in ways which do not involve sequence, or both, but which is capable of converting fats to fatty acids in about the same ratio as kid goat rennet preparations are capable of converting fats to fatty acids so that the same flavor is also imparted to the product. In one embodiment the fats are derived from a dairy product .

Another object of the present invention is to provide an isolated polypeptide wherein a polyHis-enterokinase is added to the N-terminus of the amino acid sequence of a kid goat pregastric esterase. The polyHis-enterokinase preferably comprises at least 5 histidine amino acids and is capable of increasing lipase polypeptide expression when expressed at the N-terminus of a lipase polypeptide such as SEQ ID NO: 2 or a derivative thereof. In one embodiment the polyHis- enterokinase comprises SEQ ID NO: 5.

Yet another object of the present invention is to provide a method for producing enzyme modified cheese which comprises reacting a dairy product with an isolated kid goat pregastric esterase, a recombinant kid goat pregastric esterase or a derivative of a kid goat pregastric esterase so that a mixture of fatty acids is produced that imparts to said cheese a flavor characteristic of cheese produced with a kid goat rennet preparation. Detailed Description of the Invention

The present invention relates to isolated and purified kid goat pregastric esterases and derivatives of kid goat pregastric esterases, also referred to herein as kPGE, that can be used in the manufacture of cheese to produce specific flavors by altering or modifying the fatty acid composition of the cheese . In one embodiment of the present invention the kid goat pregastric esterase is produced by recombinant technology in a non-kid or non-goat cell. The term "rkPGE" refers to recombinant kid pregastric esterase. The present invention also relates to a method of producing cheese using isolated kid goat pregastric esterase or a derivative thereof to modify the fatty acid composition of a cheese in order to impart a particular flavor to the cheese. When a rkPGE of the present invention is used in the production of the cheese, the cheese is not contaminated with other animal proteins and the product is acceptable for kosher as well as vegetarian diets. The kid goat pregastric esterases and derivatives thereof of the present invention are capable of producing a fatty acid mixture that is very similar to the mixture produced by a commercially available kid goat rennet preparation. Therefore, the present invention provides methods to produce cheese that have an enhanced flavor without use of commercial rennet. An advantage of the compositions of the present invention is that they can be used to produce cheese products acceptable for kosher and/or vegetarian diets.

For purposes of the present invention by kPGE derivative it is meant to be inclusive of kPGE polypeptides which differ from naturally occurring kPGE in amino acid sequence or in ways which do not involve sequence, or both, but which are capable of converting fats to fatty acids in about the same ratio as kid goat rennet preparations are capable of converting fats to fatty acids having the same ratio so that the same flavor is imparted to the product. kPGE derivatives of the present invention with this capability can be routinely identified by those of skill in the art in accordance with the methods taught herein. kPGE derivatives of the present invention differing in amino acid sequence from naturally occurring kPGE preferably exhibit at least 80% homology, more preferably at least 90% homology, and even more preferably at least 95% homology with SEQ ID NO: 2. Derivatives differing in amino acid sequence include polypeptides with additions, substitutions and/or deletions in their amino acid sequence as compared to SEQ ID NO: 2.

For example, kPGE derivatives are produced when one or more amino acids in naturally occurring kPGE is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid. In this embodiment, it is preferred that the derivative differ in sequence from that of SEQ ID NO: 2 by one or more conservative amino acid substitutions which typically have minimal influence on the secondary structure and hydrophobic nature of the protein or peptide. By conservative substitution it is meant the substitution of one amino acid for another with similar characteristics. For example, it is known in the art that non-polar or hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine while polar, neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Similarly, it is well known that positively charged, basic amino acids include arginine, lysine and histidine, while negatively charged acidic amino acids include aspartic acid and glutamic acid. Accordingly, some exemplary conservative substitutions for derivatives of the present invention include, but are not limited to, substitutions such as valine with glycine, glycine with alanine, valine with isoleucine, aspartic acid with glutamic acid, asparagine with glutamine, serine with threonine, lysine with arginine, and phenylalanine with tyrosine. Other conservative amino acid substitutions useful in derivatives of the present invention are set forth in the following chart obtained from Dayoff in the Atlas of Protein Sequence and Structure (1988)

Alternatively, derivatives may comprise insertions, deletions and/or substitutions which are non-conservative and which change the polypeptide to provide for certain advantages. For example, derivatives comprising an amino acid substitution which is less conservative may result in a derivative with changes in charge, conformation, and/or biological properties. Examples of such substitutions include, but are not limited to, substitution of hydrophilic residues for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain and substitution of a residue having a net positive charge for a residue having a net negative charge.

Other kPGE derivatives of the present invention include polypeptides modified to increase stability. Modifications to polypeptides which increase stability are well known. For examples, replacement of L-amino acids with D-amino acids has been shown to increase resistance of a polypeptide to proteases. See, e.g. U.S. Patent 5,219,990, which is herein incorporated by reference in its entirety. Examples of derivatives of the present invention include, but are not limited to, polypeptides identical in amino acid sequence to SEQ ID NO: 2 but with one or more non-peptide bonds and polypeptides containing residues other than naturally occurring L-amino acids, such as D-amino acids, or synthetic amino acids such as beta and gamma amino acids and cyclic derivatives .

By "derivatives" of the present invention with non- sequence modifications it is also meant to be inclusive of polypeptides comprising naturally occurring kPGE chemically derivatized either in vivo or in vi tro at select portions. For example, the naturally occurring kPGE may be acetylated, methylated, phosphorylated, carboxylated or glycosylated. Additional chemical modifications which can be made to the naturally occurring kPGE to produce a derivative of the present invention include, but are not limited to, amidation, sulfation, single or multiple halogenation, alkylation, carboxylation and phosphorylation. The kPGE polypeptide may also be singly or multiply acylated, such as with an acetyl group, with a farnesyl moiety, or with a fatty acid, which may be saturated, monounsaturated or polyunsaturated. The fatty acid may also be fluorinated. By derivative it is also meant to be inclusive of methionine analogs, such as methionine sulfone and methionine sulfoxide analogs, of the naturally occurring kPGE as well as salts of this protein. Examples of salts include, but are not limited to, ammonium salts such as alkyl and aryl ammonium salts, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, thiosulfate, carbonate, bicarbonate, benzoate, sulfonate, thionsulfonate, mesylate, ethyl sulfonate and benzenesulfonate .

By "derivative" as used herein it is also meant to be inclusive of peptidomimetics of naturally occurring kPGE. The design and production of peptidomimetics based upon a known amino acid sequence as taught herein can be performed routinely by one of skill in the art through substitution of selected R groups or amino acids in the protein with non- physiological, non-natural replacements. In a preferred embodiment, the substitutions increases the stability of the peptidomimetic beyond that of the naturally occurring kPGE. Derivatives within the scope of the present invention can be routinely identified in accordance with methods taught herein by their ability to convert fats to fatty acids in about the same ratio as kid goat rennet preparations are capable of converting fats to fatty acids having the same ratio so that the same flavor is imparted to the product.

In the context of the present invention the terms "alter" and "modify" when used in conjunction with "fatty acid mixture" are understood to mean supplying or imparting of a flavor or character to an otherwise bland, relatively tasteless substance, or augmenting an existing flavor characteristic where the natural flavor is deficient in some way, or supplementing the existing flavor to modify its organoleptic character. A kPGE of the present invention was isolated and ultra- purified from lingual tissues of kid goats. First the partial amino acid sequence and then the full amino sequence of this isolated protein was determined. The amino acid sequence of the isolated kPGE of the present invention is depicted in SEQ ID NO: 1 and 2.

In addition to production of an isolated kPGE by purifying the kPGE from kid gullet, the kPGE can be produced by recombinant genetic expression in a non-kid goat cell. The non-kid goat cell can be bacterial, fungal, yeast, or animal and can be selected by one of skill in the art based on the technology available. In a preferred embodiment, the non-kid goat cell is a yeast cell, Saccharomyces cerevisiae, or a bacterial cell, E. coli , or an animal cell, such as a Chinese Hamster Ovary (CHO) cell. A recombinant kPGE of the present invention has been produced in both yeast and E. coli . Production in a non-kid goat cell resulted in a derivative kPGE of the present invention which is glycosylated. A yeast strain comprising a recombinant DNA molecule that expresses the derivative kPGE was deposited with the Northern Regional Research Center and received deposit no. NRRL Y-30030.

Both the isolated and purified kPGE and the derivative recombinant kPGE were shown to have significant sequence homology with other well-known lipases. This homology is indicative of these kPGE having similar biological activity, i.e., the ability to convert fats to fatty acids in the same manner as commercially available kid goat rennet preparations. Experiments have been performed to demonstrate the functionality of the kPGEs of the present invention. In esterase functionality assays, kPGE samples were shown to have the ability to produce fatty acid profiles similar to those produced by commercially available goat rennet preparations. In addition, in organoleptic evaluations the isolated and recombinant kPGE samples had similar organoleptic properties to that of commercially available kid goat rennet preparations .

Using recombinant techniques, another derivative kPGE has also been identified which is capable of converting fats to fatty acids in about the same ratio as kPGE, thus modifying fatty acid composition of a dairy product in order to alter or enhance flavor. It has now been found that when a polyHis- enterokinase is added to the N-terminus of a kPGE polypeptide the kPGE derivative maintains its biological activity to convert fats to fatty acids. Further, the polyHis- enterokinase kPGE polypeptide derivative is capable of increasing lipase polypeptide expression when expressed at the N-terminus of the kPGE polypeptide. It was found that the polyHis-enterokinase had greatest activity when comprising at least 5 His amino acids (SEQ ID NO. 5) . The present invention also provides polynucleotides capable of expressing kPGE polypeptides and derivatives of the present invention. In a preferred embodiment, the polynucleotides encodes a kPGE or a derivative polypeptide comprising SEQ ID N0:2. An exemplary polynucleotide is depicted in SEQ ID NO:l and SEQ ID NO: 3. Polynucleotides of the present invention may comprise DNA or RNA. The polynucleotide may further comprise a nucleotide sequence encoding a polyHis-enterokinase polypeptide. An exemplary polyHis enterokinase peptide is depicted in SEQ ID NO: 5 and an exemplary polynucleotide sequence encoding this peptide is depicted in SEQ ID NO: 6.

The present invention also relates to a transforming nucleic acid molecule comprising a plasmid or vector containing a nucleic acid sequence encoding the amino acid sequence of kPGE or a derivative polypeptide. In a preferred embodiment the transforming nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 4.

Therefore, another aspect of the present invention relates to a process for recombinant production of a kPGE by isolating a polynucleotide encoding a kPGE, inserting the isolated polynucleotide into a vector or plasmid suitable to transform a host cell, transforming a host cell with the vector or plasmid comprising the isolated polynucleotide, and growing the transformed cells so that the kPGE in recombinant form is expressed.

In reacting the isolated or recombinant kPGE of the present invention, or the polyHis-enterokinase polypeptide derivative, with a dairy product, a mixture of fatty acids is produced. In the context of the present invention the dairy product may comprise, but is not limited to, lipolyzed butter oil, milk, cheese, and/or whey. Therefore, the present invention also provides a method for producing a mixture of fatty acids from a dairy product so that a particular flavor is produced, wherein the flavor produced is characteristic of the flavor produced from naturally-occurring kid goat pregastric esterase in a commercial rennet preparation. Thus, the isolated kPGE or recombinant kPGE or derivative kPGE of the present invention can be used in the production of enzyme modified cheeses or EMCs and as a substitute for commercial rennet preparations or in addition to such commercial rennet preparation during cheese production. Methods for production of cheeses with flavor enhancements based on the presence of certain fatty acid mixtures is well known in the art. Reviews of such methods can be found in texts such as Godfrey and West (1996. Flavor Production wi th Enzymes . Industrial Enzymology. 2^nd edition, Stockton Press; Chaudhari, R.V. and G.H. Richardson. J. Dairy Sci . 1970. 54:467-471.

The kPGE protein of the present invention can be expressed in microorganisms and transgenic animals through recombinant technology. A recombinant kPGE then can be fixed and delivered into food systems by spray drying or encapsulation. The microbial production of the enzyme will allow for development of new kosher and vegetarian food products . The following non-limiting examples are provided to further illustrate the present invention.

EXAMPLES

Example 1: Purification of kPGE Protein

A 250 ml 2.5 x 50 cm Bio-Rad Econo column was packed with approximately 220 ml Bio-Rad Exchange Q chromatography matrix as specified by the supplier (Bio-Rad Corporation,

Hercules, CA) . The column was washed and equilibrated in 50 mM Tris-HCl buffer, pH 8.0.

Five grams of Aurotech Kid Pregastric Esterase (390 Ramsey Units) were brought up in 50 mM Tris-HCl, pH 8.0, mixed by stirring for approximately 20 minutes and centrifuged at

6500 rpm for 10 minutes. The supernatant was decanted and recentrifuged at the same speed. The recovered supernatant

(80 ml) was loaded onto the column described above, 2 ml/min flow rate.

The column was washed with buffer for 100 minutes with an initial flow rate of 2 ml/min that changed to 1.5 ml/min by the end of the period. At 100 minutes, a 100 minute linear gradient from 100% original buffer to 100% new buffer (1 M NaCl, 50 mM Tris-HCl, pH 8.0) was begun. After 25 minutes, fraction collection began and continued for 124 minutes. At 200 minutes, the gradient was held at 100% new buffer for 50 minutes before switching to 100% original buffer and then was held 200 minutes for re-equilibration of the column.

Activity of the fractions for kPGE activity was assayed at 405 nm using p-nitrophenol butyrate substrate. Sample (20 μl) were placed in microtiter dish wells and diluted with 180 μl of substrate solution (30 mg p-nitrophenol butyrate dissolved in 10 ml isopropanol, then 1 ml added to 9 ml 4.4% Triton X-100, 0.11% Gum Arabic, 50 mM Tris-HCl, ph 8.0).

Fractions found to contain kPGE activity were pooled, diafiltered with 20 mM BIS-TRIS (bis [2-hydrosyethyl] imino- tris [hydrosymethyl] methane) buffer, pH 7.1, and then loaded onto a column containing 200 ml PBE 94 chromatography gel

(Pharmacia Biotech, Inc., Piscataway, NJ) for chromatofocusing in the pH range of 9-4. The column was developed with a 1:10 dilution of Polbuffer 74 (Pharmacia Biotech, Piscataway, NJ) pH 4.0. Fractions were collected and assayed as described above for kPGE activity. Fractions found to contain kPGE activity were again pooled, concentrated, and diafiltered against distilled water using a stirred cell device (Amicon, Inc., Beverly, MA), fitted with a high-flow, inert non-ionic membrane retaining 90% of molecules with molecular masses greater than 30,000 Daltons (PM30, Amicon, Inc., Beverly, MA).

The resulting partially-purified and concentrated kPGE was subjected to electrophoresis in precast 12% polyacrylamide gels containing 375 mM Tris-HCl buffer, pH 8.8 (Bio-Rad Laboratories, Hercules, CA) to separate protein species from one another. Following separation, the kPGE protein species was localized to specific regions of the gel by making horizontal cuts along the length of the gel (1 mm cuts) . This resulted in a continuous series of 1 mm segments that contained protein species that had migrated at similar rates to end up in the same relative position in the gel. A small piece of each individual segment was macerated in Tris-HCl buffer, pH 8.0, and assayed for kPGE activity using the substrate assay described above. Those acrylamide segments showing kPGE activity were then macerated in buffer and subjected to electrophoresis in an electroelution device (Isco, Inc., Lincoln, NE) . In this way, kPGE activity was electroeluted and concentrated in buffer. KPGE activity was reconfirmed and electrophoresed in sodium dodecyl sulfate (SDS) to demonstrate recovery of a 50,000 Dalton protein species. In addition, traditional Ramsey Unit assays were conducted to verify that classical kPGE activity was recovered. This assay follows the rate of change of pH that results from lipase acting on tributerin to release butyric acid. Combined lots of the resulting isolated and ultra- purified kPGE were assayed for esterase functionality, i . e . , flavor modification.

Example 2 : Determination of the Partial Amino Acid Sequences of kPGE and Demonstration of Homology Following native gel electrophoresis, proteins in the polyacrylamide gel were electrophoretically transferred to a polyvinylidenedifloride (PVDF) membrane support using a Western blot procedure. Following transfer, staining of the PVDF membrane allowed detection of the ultra-purified kPGE protein as a unique band. The band was subjected to N- terminus amino acid sequencing to yield a partial N-terminus sequence. Multiple recoveries of similarly purified kPGE bands were also subjected to protease digestion to release specific kPGE peptide fragments and the resulting fragment mixture was subjected to HPLC for separation of fragments.

Individual fragments were then subjected to N-terminus amino acid sequencing to obtain sequence data for three additional fragments internal to the kPGE protein. A search of the National Biomedical Research Foundation (NBRF) protein database using the partial amino acid sequences led to identification of high homology within regions of the human gastric lipase and rat lingual lipase.

Example 3 : Isolation of RNA and Construction of cDNA

Library of Cloned Sequences from Goat Lingual Tissues

Frozen kid lingual tissue (parotid salivary glands and sublingual tissues of kid goat tongue) was homogenized in a lysis buffer (Tris-HCl pH 8.0, LiCl, EDTA, Li dodecyl sulfate and dithiothreitol) polyadenylated messenger RNA (polyA-mRNA) using a commercial product, Dynabeads Oligo (dT)25 (Dynal, Inc. Lake Success, NY) . Purified polyA-mRNA was primed with an oligonucleotide consisting primarily of poly- deoxythymidine DNA and reverse transcribed into DNA using reverse transcriptase. The resulting double-stranded DNA molecules were then cut into Eco Rl restriction enzyme and ligated into EcoRl-cut Lambda ZAP II vector DNA (Stratagene Cloning Systems, La Jolla, CA) to produce a library of Lambda ZAP II DNAs, each of which presumably contained one cDNA derived from one mRNA that was present in the kid goat lingual tissue mRNA population. The library of cDNA- containing Lambda ZAP II DNAs was packaged to form virulent bacteriophage using a commercially prepared packaging system (Gigapack II gold packaging extract, Stratagene Cloning Systems, La Jolla CA) and used to infect an appropriate strain of bacteria (XLl-Blue, Stratagene Cloning Systems, La Jolla, CA) .

Example 4: Development of Oligonucleotide Probes for Recognition and Recovery of the kPGE Gene

Using the kPGE amino acid sequences determined, synthetic oligonucleotides were designed to be used in generating fragments of DNA that represent parts of the kPGE gene. Certain regions of the partial amino acid sequences were reverse translated into corresponding DNA sequences to act as primers using PCR. PCR techniques allowed synthesis and amplification of regions of DNA that lay between the two primers. Since the lipases as a class, including the pregastric esterases, are similar in size, approximately 50,000 Daltons, and the relative regions of homology of the partial kPGE amino acid sequences were known, the relative size of the expected DNA fragment from PCR analysis with any specific primer could be predicted. Thus, specific kPGE- based primers, conserved lipase-based primers or combinations thereof were used to carry out PCR using the library- containing bacteriophage to generate specific DNA fragments. Several of the combinations yielded DNA fragment sizes expected to result from authentic kPGE gene sequences, but did not yield correctly sized DNA fragments DNA was used. In this way, the library was shown to contain DNA sequences of kPGE-like genes. DNA fragments resulting from these PCR amplifications were cloned into a plasmid vector, pT7Blue T- vector (Novagen, Inc., Madison, WI) and transformed into bacterial cells (Novablue competent E. coli from Novagen Inc., Madison, WI) using well-known bacterial transformation procedures. Plasmid DNA preparations were made from several different transformed clones to recover larger quantities of purified DNAs containing different kPGE-like gene fragments. Several of these were DNA sequenced using common techniques and one was selected (GS 1972) as clearly containing DNA sequence that when translated would produce a protein with high amino acid sequence homology to comparable regions of other pre-duodenal and lingual lipases.

Example 5: Identification of the Cloned kPGE Gene

Plasmid DNA from bacterial clone GS 1972 was shown to consist of plasmid vector pT7Blue T-vector (Novagen, Inc., Madison, WI) with an integrated PCR-generated DNA fragment (441 base pairs) corresponding to the translated region of amino acid residue 18 to 164 of other known, mature pre- duodenal and lingual lipases. This purified DNA was radioactively labeled with S³⁵ and used to identify phage carrying cDNAs with homologous regions using common screening procedures. Since the primary phage library contains millions of phage in a highly concentrated form, several rounds of phage purification were conducted to separate the phage of interest, those containing kPGE-like cDNAs, from all others. Thus, semi-purified phage preparations were first identified by diluting the phage and plating on agar such that single phage plaques were clearly identified. Replicas of the phage plaque patterns that occurred on agar plates were transferred to nitrocellulose membranes and probed to identify phage plaques of interest. Phage were then taken from the positive plaques on the agar plates and used to identify those that yielded a 440 base pair fragment when amplified using the PCR primers. Following initial identification of 10 positive semi-purified phage preparations, secondary and tertiary screens were performed to result in identification of 5 highly purified phage preparations that yielded a hybridizing signal when labeled with radioactive plasmid DNA from clone GS 1972 and a 440 base pair fragment when amplified using the original primers in PCR.

XL1 Blue cells (Stratagene Cloning Systems, La Jolla, CA) were infected with the 5 purified phage preparations along with M13 helper phage to convert the cloned fragments from a phage form into a plasmid form. Proteins produced by the M13 helper phage cut the phage DNA on one side of the cloned insert DNA and replicated the DNA through to the other side. The smaller, newly synthesized single-stranded DNA then circularized, was packaged, and secreted from the cell. The secreted plasmid was used to transform SOLR bacterial cells (Stratagene Coning Systems, La Jolla, CA) along with another helper phage, VCSM13, to convert the plasmid into a stable, replicating plasmid. Four E. coli strains were thus obtained that contained pBluescript SK-double stranded plasmids with cloned cDNA inserts of interest. DNA sequencing of the cDNA inserts of the plasmids yielded a nucleotide sequence, a portion of which translated into a PGE-like enzyme. The translated sequence was comprised of 378 amino acids that form a protein with a calculated molecular mass of 42,687 Daltons. By comparison, human gastric lipase has 378 amino acids and a calculated molecular mass of 43,208 Daltons ; bovine pregastric esterase has 378 amino acids and a calculated molecular mass of 42,987 Daltons; while rat lingual lipase has 376 amino acids and a calculated molecular mass of 42,700 Daltons. A comparison of the amino acid sequence alignments indicated the similarity among these enzymes. At the DNA level, strong homology was also apparent. Inspection of the translated sequence of the PGE-like gene confirmed the presence of amino acid sequences that were determined from the purified kPGE enzyme, confirming the recovery of the kPGE gene.

Example 6: Esterase Functionality Assay

All esterase samples had been received frozen after storage at -18°C. The positive control for the assay was original kid goat lipase, 390 U/gram. The negative control for the assay was 0.6 ml of 20 mM phosphate buffer, pH 7.0. Samples were diluted in 20 mM phosphate buffer, pH 7.0 (0.6 ml) . The substrate was 40% fat cream obtained from Golden Guernsey Dairy. This cream was free of added mono- and di- glyceride. Usage level of all lipase samples was 0.78 U/gram of cream, which was comparable to the usage level in cheese production. Substrate (3.8 gram) was added to 10 ml plastic tubes. The sample (0.6 ml) was then mixed with the substrate . Tubes were incubated for 72 hours . At the end of incubation, each sample was titrated. The free fatty acid profile of each sample was analyzed. In this method the following fatty acids were quantified: butyric, hexanoic, octanoic, decanoic, lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic, and linolenic acids. Each sample was analyzed twice and the results were averaged.

Total free fatty acids released by each sample tested were different even though the same amount of activity units were used in the incubation sample 1: titration level of 3.3; sample 2: titration level of 2.4) . The control lipase had the highest activity (titration level of 5.4). The negative control titration level was 1.7.

Free fatty acid (FFA) profiles (percent of FFA) are shown in the following Table 1. The results are reported as percent of free fatty acid (FFA) . The data indicated that the positive control sample showed a typical profile of milk lipase. Sample 1 had a profile almost identical to the positive control. Sample 2, however, had a different profile with a much lower percentage of short chain fatty acids and a higher percentage of long chain fatty acids. The overall activity of sample 2 was also much lower. This change in sample profile may be due to the impact of milk lipase on the system, where low lipase activity led to a larger impact of milk lipase.

An organoleptic analysis was performed. A cheese sauce consisting of margarine, modified starch and VELVEETA cheese and water was used as the base for evaluation as it is routinely used in such circumstances. Since the samples had different titration levels, the usage level of each sample was varied to compensate for titration variations. Overall, all the samples tested showed similar organoleptic properties. They were not identified as typical fatty acid and had a culture milk type of flavor.

Example 7: Extraction and Analysis of Free Fatty Acids for Dairy Products

This procedure extracts free fatty acids from lipolyzed butter and EMCs (enzyme modified cheeses) . The extract is then analyzed by gas chromatography. This procedure is adapted from Deeth, H.C. et al . 1983. New Zealand Journal of Dairy Science and Technology 18:13-20. This procedure has been extensively tested for extraction efficiency. The adjustment for this procedure is the sample size which depends on the amount of free fatty acids in the sample.

A bonded phase capillary tube was used to give a superior chromatogram compared to Deeth et al., especially for long chain fatty acids. Heptanoic acid was used as an internal standard for fatty acids with chain length of up to 10 carbons, while pentadecanoic acid was used for fatty acids with chain lengths of more than 12 carbons. The following reagents were used: necessary free fatty acids, isopropyl ether (99%), diethyl ether (99.9%; spectrophotometric grade), hexane (spectrophotometric grade) , formic acid (96%; ACS reagent) , activated aluminum oxide (acidic, Erockznan I) , 4N sulfuric acid, and glass wool treated with phosphoric acid. The procedure used a three level calibration (designated as Levels 1, 2 and 3 in Table 2 below) for each fatty acid peak. The procedure cannot quantify acetic acid in the product because formic acid used in the procedure contains small amounts of acetic acid which interferes with quantifying the acetic acid extracted from the sample.

The following fatty acids were weighed directly into a 100 ml volumetric flask:

It is recommended that weighing starts with the longer chain standards because they are less volatile, minimizing loss due to evaporation. Four grams of formic acid were then added into each flask. Each flask was filled to the 100 ml mark with isopropyl ether and mixed well. Standard were stored in small glass bottles (10 ml) with chemical resistant caps in a freezer at -20° C.

The internal standard, containing heptanoic acid and pentadecanoic acid, was prepared by weighing 0.3 grain of heptanoic acid and pentadecanoic acid into a 100 ml volumetric flask and then filling the flask with room temperature hexane to the 100 ml mark. The standard was then mixed well and stored in small glass bottles (10 ml) with chemical resistant caps in a refrigerator. This standard must be used at room temperature.

Column packing was prepared by mixing 4% water with deactivated alumina. The column packing was then allowed to dry and equilibrate overnight in a desiccator. Free fatty acids were then extracted from samples. For samples with low degree of lipolysis, such as samples with about 4 ml 0.05 N NaOH free fatty acid titration per gram, an approximately 0.3 gram sample was used. For samples with strong lipolysis, such as samples with free fatty acid titration of 16 ml 0.05 N NaOH per gram, an approximately 0.1 gram sample size was used.

A solvent mixture for the extraction was prepared that contained hexane/diethyl ether at a ratio of 1:1 (vol:vol). Enough solvent was prepared to assure that extraction could be completed, approximately 35 to 40 ml. The sample to be extracted was weighed into a 50 ml screw cap centrifuge tube. Next, 0.1 ml of 4 N sulfuric acid was added into each tube. The internal standard solution (0.1 ml) was added into each tube. The mg of internal standard (ISTD) added per tube was calculated by the following formula: mg ISTD/g sample = 0.1 x concentration ISTD (mg/ml)

Sample size (g)

Anhydrous sodium sulfate (1 g) was added into each tube. Then, 10 ml of the extraction solvent was added into each tube, and the tubes were capped and mixed on a vortex mixer at the highest setting. The extraction proceeded for 30 minutes to 1 hour. For lipolyzed butter samples which dissolve in extraction mixture easily, a 30 minute extraction was sufficient. In the case of EMCs, an extraction time of up to 1 hour was needed. The tubes were then centrifuged at 7000 rpm (0°C) for 5 minutes which resulted in a clear supernatant that contained free fatty acids and fat .

A glass column was packed with deactivated alumina and the supernatant was introduced to the column. After all the extract had passed through the column, the column was washed with the collected filtrate to give the supernatant a second column pass. The column was washed with 5 ml of the hexane/diethyl ether mixture two times. The column was dried under vacuum until the packing inside was a free flowing powder. The alumina was transferred into a small vial and capped. This procedure was repeated for each sample with a new alumina column.

A solution of 6.0% formic acid with isopropyl ether was prepared to release the fatty acids from the alumina packing. A 0.5 gram sample of the alumina was weighed into a disposable micro centrifuge tube (1.5 ml) and mixed with 0.5 ml formic acid solution. The tube then stood for 30 minutes with occasional mixing. The tube was centrifuged for 2 minutes to obtain clear supernatant, the supernatant transferred to a 1 ml vial and then subjected to gas chromatographic (GC) analysis.

The extract was analyzed by GC using the following conditions: 1) column, HP-FFAP, 25 M X 32 mm with 0.52 micron film thickness; 2) guard column, Restek capillary guard column of 5 M x 0.32 mm; 3) injector temperature, 28°C; 4) detector temperature, 30°C; 5) oven temperature, 100 C° to 240°C at 8°C/minute; 6) initial isothermal time, 0 minutes; 7) final isothermal time, 12.5 minutes; 8) total analysis time, 30 minutes; 9) initial inlet pressure, 20.0 psi; 10) constant flow, ON; 11) flow rate, 3.7 ml/min; 12) split flow, 20 ml/min; 13) detection, FID; and 14) injection, 0.2 microliter . The amount of free fatty acid per gram of product was then calculated. The three levels of standards (1, 2 and 3) were analyzed with the same GC program. A calibration table was built containing three level linear calibration for each peak. In order to calculate the Mol% (Mol% is useful for recognizing a fatty acid profile) , the mmole of each fatty acid per gram sample was determined. This was done by dividing the amount of each fatty acid (mg/g sample) by its molecular weight. The sum of all the calculated mmole of each fatty acid per gram sample was determined to obtain the total mmole of free fatty acid per gram of sample. The mmole of each fatty acid per gram sample was then divided by the total mmole of free fatty acid per gram of sample. Finally, the calculated value was multiplied by 100 to give the Mol%.

Claims

What is claimed is;

1. An isolated kid goat pregastric esterase comprising SEQ ID NO: 2, said isolated kid goat pregastric esterase being free of other kid goat proteins .

2. The isolated kid goat pregastric esterase of claim 1 wherein said isolated kid goat pregastric esterase is produced by purification from tissues of the kid goat gullet.

3. The isolated kid goat pregastric esterase of claim 1 wherein said esterase is produced by recombinant expression in a non-kid goat cell.

4. The isolated kid goat pregastric esterase of claim

3 wherein the non-kid goat cell comprises a bacterial cell, a fungal cell, a yeast cell or an animal cell.

5. The isolated kid goat pregastric esterase of claim 4 wherein the cell is a yeast cell.

6. The isolated kid goat pregastric esterase of claim 5 wherein the yeast cell is Saccharomyces cerevisiae.

7. The isolated kid goat pregastric esterase of claim 5 encoded by a transforming nucleic acid molecule comprising SEQ ID NO: 4.

8. The isolated kid goat pregastric esterase of claim

4 wherein the cell is a bacterial cell.

9. The isolated kid goat pregastric esterase of claim 7 wherein the bacterial cell is E. coli .

10. The isolated kid goat pregastric esterase of claim 1 encoded by a polynucleotide comprising SEQ ID NO: 3.

11. An isolated polypeptide comprising a derivative of the isolated kid goat pregastric esterase of SEQ ID NO: 2, said derivative converting fats to fatty acids in about the same ratio as kid goat rennet preparations convert fats to fatty acids.

12. The isolated polypeptide of claim 11 wherein the derivative is 80% homologous with SEQ ID NO: 2.

13. The isolated polypeptide of claim 11 wherein the derivative comprises a glycosylated isolated kid goat pregastric esterase.

14. The isolated polypeptide of claim 11 wherein the derivative comprises a polyHis-enterokinase at an N-terminus of its amino acid sequence.

15. The isolated polypeptide of claim 14 wherein the polyHis-enterokinase comprises SEQ ID NO: 5.

16. The isolated polypeptide of claim 14 wherein the polyHis-enterokinase is at the N-terminus of SEQ ID NO: 2.

17. A method for producing enzyme modified cheese comprising reacting a dairy product with an isolated kid goat pregastric esterase, a recombinant kid goat pregastric esterase or a derivative of a kid goat pregastric esterase so that a mixture of fatty acids is produced that imparts to said cheese a flavor characteristic of cheese produced with a kid goat rennet preparation.

18. The method of claim 17 wherein the dairy product is lipolyzed butter oil, milk, cheese or whey.

19. The method of claim 17 wherein said isolated kid goat pregastric esterase comprises SEQ ID NO: 2.

20. The method of claim 17 wherein said derivative of kid goat pregastric esterase is 80% homologous with SEQ ID NO : 2.

21. The method of claim 20 wherein said derivative of kid goat pregastric esterase further comprises SEQ ID NO: 5.