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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• HASHs halogenated aliphatic hydrocarbons
• HAHs produced as co-products in chemical manufacturing process can be burned to produce heat and, in some cases, be recycled to low value starting materials, thus yielding some recovery from a waste product or excess co-product.
• HAH degradation occurs by microbial biodegradation.
• Biodegradation of HAHs results in the formation of carbon dioxide, water, and hydrochloric acid when the halogen is a chloride.
• the biodegradation of HAHs to carbon dioxide, water, and hydrochloric acid by select microorganisms is disclosed in U.S. Patent Nos. 4,853,334 and 4,877,736.
• 5,372,944 discloses a Rhodoccocus species which produces a dehalogenase which converts HAHs to haiohydrins.
• these references largely rely on cellular systems and do not take advantage of the benefits that may be obtained from the use of an immobilized, activity-modified enzyme in a continuous feed process.
• U.S. Patent No. 5,372,944 relies on Rhodococcus cultures comprising wild type or mutant cells.
• the mutation techniques taught therein do not take advantage of recombinant DNA methods and so fail to capitalize on the benefits these methods offer in terms of improvement in activity and expression of the dehalogenase enzyme.
• HAHs Rather than depend on biodegradation of HAHs by cell cultures, it would be advantageous to have an improved, recombinant enzyme that can be readily adapted to continuous-feed methods whereby the HAHs could be efficiently converted to valuable intermediates for use in production of other useful products, such as chemical intermediates in the preparation of polyethers to form polyurethanes or in the preparation of glycols and polyglycols to form lubricants, surfactants, emulsifiers, etc.
• the present invention is directed to a recombinant enzyme, capable of converting HAHs to vicinal halohydrins, comprising an amino acid sequence substantially homologous with the amino acid sequence of residues 1 -292 of Figure 2.
• Another object of the invention is to provide DNA sequences encoding a polypeptide comprising such an enzyme, more specifically to DNA sequences comprising a polynucleotide substantially homologous with the nucleotide sequence of bases 37-912 of Figure 2.
• Another object of the invention is to provide a vector containing the DNA sequence(s) and a method for producing the polypeptide comprising placing the vector into a host cell and growing the host cell under conditions allowing the transformant to produce the dehalogenase.
• Further objects of the present invention are to provide an immobilized form of the enzyme on a solid support as well as a process for converting a HAH to an alcohol or halohydrin comprising contacting the HAH with the immobilized enzyme.
• FIG. 1 illustrates a plasmid map of the vector pEXPROK.
• Plasmid pEXPROK is derived from the commercially available pPROK-1 plasmid (Clontech, Mountain View, CA) containing the Ptac promoter and the 5S, T1T2 terminator sequences. In the figure, the T1T2 region is indicator by "Term.”
• This plasmid was generated by replacing the pPROK-1 multiple cloning site with a pair of oligonucleotides which introduced restriction site Nco I, Hind III, Xho I, Nhe I, and Not I into the linker.
• the "ATG" sequence of the Nco I site represents a functional in-frame start site.
• the Nhe I site is followed by the EXFLAG linker sequence.
• the sequence of the EXFLAG linker corresponds to nucleotides 919-975 in Figure 2 and encodes amino acids 295-315 in the RDhl protein sequence shown
• Figure 2 presents the nucleotide sequence encoding the putative Rhodococcus rhodochrous TDTM003 haloalkane dehalogenase enzyme and the amino acid sequence derived from this nucleotide sequence.
• Amino acid residues 1 -292 correspond to the Rhodococcus dehalogenase (RDhl) structural gene and are encoded by nucleotides 37-912.
• Amino acid residues -12 through -1 represent a polyhistidine-containing amino-terminal tail, with residues -12 and -11 participating in the formation of both the translational start site and the Nco I cloning site.
• Amino residues 293- 294 are encoded by the Nhe I cloning site and are followed by amino acids 295-305, which are referred to herein as the EXFLAG peptide.
• the EXFLAG linker (nucleotides 919-975) encodes the EXFLAG peptide and a dual-translational stop site (each indicated by an asterisk).
• Figure 3 illustrates a plasmid map of the vector pEXPROK-RDhl.
• Figure 4 presents an alignment comparison chart of the amino acid sequences of the putative Rhodococcus rhodochrous TDTM003 haloalkane dehalogenase, the Xanthobacter autotrophicus GJ10 dehalogenase, the Renilla reniformis luciferin monooxygenase, and the Pseudomonas spp. LinB gene product (a tetrachloro- cyclohexadiene hydrolase).
• Figure 5 presents a plasmid map of the vector pRDhl-KO2.3-EXPROK comprising the putative Rhodococcus rhodochrous TDTM003 haloalkane dehalogenase gene under the control of the IPTG-inducible Ptac transcription promoter.
• Figure 6 illustrates a plasmid map of the high level expression vector pRSET-RDhl comprising the putative Rhodococcus rhodochrous TDTM003 haloalkane dehalogenase gene under the control of the T7 transcription promoter.
• Figure 7 illustrates a plasmid map of the high level expression vector pTrcHis-RDhl comprising the putative Rhodococcus rhodochrous TDTM003 haloalkane dehalogenase gene under the control of the trc transcription promoter.
• Figure 8 illustrates a plasmid map of the high level expression vector pTrxFus-RDhl comprising a modified version of the putative Rhodococcus rhodochrous TDTM003 haloalkane dehalogenase gene fused to the gene encoding E. coli thioredoxin, the combined fusion gene being under the control of the P L transcription promoter.
• Figure 9 presents an image of an SDS-PAGE gel of cell lysate samples from cells expressing the pEXPROK-RDhl clone, compared to the partially purified rRDhl enzyme.
• Figure 10 presents an image of an anti-FLAG antibody immunoblot of an SDS-PAGE gel identical to that of Figure 9.
• Figure 1 1 presents an image of an SDS-PAGE gel of cell-free extracts from cells expressing pRSET-RDhl.
• Figure 12 presents an image of an anti-FLAG antibody immunoblot of an SDS-PAGE gel identical to that of Figure 1 1.
• Figure 13 presents an image of an SDS-PAGE gel of cell-free extracts from cells expressing pTrcHis-RDhl.
• Figure 14 presents an image of an anti-FLAG antibody immunoblot of an SDS-PAGE gel identical to that of Figure 13.
• Figure 15 presents an image of an SDS-PAGE gel of cell-free extracts from cells expressing pTrxFus-RDhl.
• Figure 16 presents a productivity profile for an immobilized enzyme bioreactor acting on the substrate, 1 ,2,3-Trichloropropane.
• Figure 17 presents a bar chart of the activities of EPPCR-mutated Rhodococcus rhodochrous haloalkane dehalogenases.
• Figure 18 presents a bar chart of the activities of EPPCR-mutated Rhodococcus rhodochrous haloalkane dehalogenases.
• Figure 19 presents a graph of enzyme activity data for an RDhl enzyme bearing a carboxy-terminal S-Tag polypeptide tail and for an RDhl enzyme bearing a carboxy-terminal EXFLAG polypeptide tail.
• the present invention results from intensive research into obtaining a DNA sequence encoding a polypeptide having haloaliphatic dehalogenase activity from a microorganism belonging to the genus Rhodococcus, making recombinant DNA sequences by integrating the DNA sequence per se - or as modified - into a vector, and transforming a microorganism with the recombinant vector. Transformants were screened for dehalogenase activity levels and from those with heightened activity, the dehalogenase enzymes were isolated. Various solid support immobilization systems were then evaluated to identify enzyme-support combinations in which the enzyme could effectively convert halogenated aliphatic hydrocarbons to alcohols or halohydrins.
• Halogenated aliphatic hydrocarbons (HAHs) subject to conversion using the immobilized dehalogenase include C 2 -C 10 aliphatic hydrocarbon molecules and groups which have two or more halogen atoms attached, wherein at least two of the halogens are on adjacent carbon atoms.
• Preferred HAHs are saturated hydrocarbons in which at least one of the halogens occupies a primary position on the molecule or group; more preferred are those in which no more than 1 halogen occupies the same carbon atom.
• HAHs are saturated hydrocarbons comprising 1 ,2-dihalo groups, examples of which are the 1 ,2-dihaloethane, 1 ,2-dihalopropane, 1 ,2-dihalobutane, and 1 ,2,3- trihalopropane molecules and groups.
• These classes include, for example, 1 ,2- dichloroethane, 1 ,2-dichloropropane, 1 ,2,-dichlorobutane, 1 ,2,3-trichloropropane, and 1 ,2- dibromo-3-chloropropane molecules and groups.
• halogen means chlorine, bromine, or iodine.
• the preferred halogens are bromine and chlorine.
• the most preferred halogen is chlorine and among the most preferred HAHs are volatile chlorinated aliphatic hydrocarbon (VCAH) molecules and groups; especially preferred VCAHs include 1 ,2-dichloropropane and 1 ,2,3- trichloropropane molecules and groups.
• halohydrin means a vicinal halohydrin, i.e. any aliphatic organic compound, other than a carboxylic acid, which contains both a hydroxyl substituent and a halogen substituent on adjacent carbon atoms of the molecule.
• ⁇ , ⁇ -halohydrins are the most preferred vicinal halohydrins.
• immunoblot and “immunoblotting” are used herein to denote the process of: 1 ) transferring protein(s) from an electrophoresis gel, e.g., a polyacrylamide gel for use in PAGE, to a protein-binding membrane; and then 2) probing that membrane with an antibody specific to protein constituents that may be included among those transferred to the membrane; and then 3) determining the location of that antibody using any of various chromogenic methods well known in the art, e.g., developing color in a colorable marker which is directly or indirectly linked to the antibody.
• An example of an immunoblotting method is the Western blot.
• permeablize permeablizing
• permeablization permeablization
• sonicate is used herein to denote the use of sonic waves to rapidly vibrate the contents of a test tube or other container, in order to thoroughly mix them.
• vortex is used herein to denote the action of mechanically gyrating a test tube along its bottom while manually holding the top of the test tube stationary, in order to mix its contents.
• selectable means “able to be selected.”
• selectable marker or “dominant selectable marker” indicates a genetic feature, such as a gene encoding an antibiotic resistance enzyme, whose presence allows the gene's host cell to multiply in a corresponding selection medium, e.g., a growth medium containing that antibiotic.
• a genetic feature such as a gene encoding an antibiotic resistance enzyme, whose presence allows the gene's host cell to multiply in a corresponding selection medium, e.g., a growth medium containing that antibiotic.
• expression construct denotes a plasmid, virus, virion, viroid, transposable element, cos-construct, transfectable carrier-associated DNA strand (e.g., a DNA-coated "gene-gun” pellet or DNA-coated natural or synthetic histone-like particle), or other DNA-to-cell delivery system which is known in the art.
• G g Glycine (Gly) S, s Serine (Ser)
• @ At, e.g., @ 37°C is “at 37°C” and @60min. is “at 60 minutes"
• a Absorbance, e.g., A 280 is "absorbance measured at 280nm" aa Amino acid
• ⁇ Change or difference e.g., ⁇ A is "change in absorbance" dATP Deoxyadenosine triphosphate DCB 1 ,4-Dichlorobutane DCH 2,3-Dichloro-1 -propanol dCTP Deoxycytidine triphosphate
• Ig Immunoglobulin e.g., IgG is "immunoglobulin G" IPTG Isopropylthiogalactopyranoside
• NP-40 Nonoxynol; p-(n-C 9 H 19 )-C 6 H 4 -(OCH 2 CH 2 ) n OH; also called nonylphenoxypolyethoxyethanol (a non-ionic detergent surfactant)
• OD Optical density e.g., OD 600 "optical density measured at 600nm” oligo Oligonucieotide p Plasmid, e.g., pRSET, pTrcHis, pTrxFus, or pUC
• RDhl Rhodococcus haloalkane dehalogenase enzyme residue An amino acid which is part of a polypeptide rpm Rotations per minute rRDhl Recombinant Rhodococcus haloalkane dehalogenase enzyme
• the dehalogenase for use in the present invention is preferably derived from Rhodococcus species ATCC 55388 and is capable of converting a HAH to a halohydrin or alcohol, preferably a halohydrin.
• the preferred recombinant enzyme comprises an enzymatically active polypeptide comprising the minimal functional portion of the wild type dehalogenase enzyme, i.e. the smallest possible segment thereof which, after proper folding, retains haloalkane dehalogenase activity.
• this polypeptide is substantially homologous with the amino acid sequence of residues 1 -292 of Figure 2.
• this polypeptide is at least about 90% homologous, even more preferably at least about 95% homologous, and yet more preferably at least about 99% homologous therewith.
• enzymatically active polypeptides having the amino acid sequence of residues 1-292 or of residues of Figure 2.
• the preferred recombinant enzyme may also comprise one or more other units such as labels, tags, tails, linkers, solid supports, chelants, other enzymes, and so forth - regardless of their size - which may either be produced with or linked to the enzymatically active polypeptide of the enzyme after it is formed. Such units may be excised from the enzyme after it has been properly folded and/or immobilized upon a solid support.
• the enzyme is produced with or linked to a substantially hydrophilic tail.
• This tail may be a hydrophilic oligopeptide expressed as part of the enzyme or may be, e.g., an oligosaccharide moiety attached by the host cell to the core enzyme after expression thereof.
• a preferred tail is a substantially hydrophilic oligopeptide expressed as part of the enzyme. More preferably, the enzyme is expressed with a highly hydrophilic oligopeptide tail. Most preferably, the oligopeptide tail is expressed at the carboxy terminus of the enzyme.
• a most preferred oligopeptide tail is a hydrophilic, carboxy-terminal tail which is rich in histidine and/or aspartic acid residues, especially one which is from about 5 to about 25 amino acids in length and contains at least about 25% histidine or aspartic acid residues, more preferably at least about 50% of such residues.
• the recombinant enzyme is preferably produced by a host cell containing at least a section of a polynucleotide having the nucleotide sequence of bases 37-912 of Figure 2.
• the present invention is also directed to recombinant DNA sequences capable of expressing the enzymes of the present invention.
• DNA sequences include those able to express the novel haloalkane dehalogenase(s) by means of translation systems not following, or not fully following, the standard DNA code's codon-to-amino acid correspondence pattern. Such systems include those in which certain codons are "suppressed" relative to the standard DNA code.
• At least one of the 20 or so amino-acid-specific classes of aminoacyl-tRNA (“aa- tRNA”) molecules contains at least one tRNA molecule - having an anticodon belonging to that class - which is linked to the "wrong” amino acid, so as to predispose the translation system to produce a "violation" of the standard DNA code (i.e. by causing the insertion, in at least one position in the growing polypeptide chain, of an amino acid not normally found in correspondence with the mRNA codon governing that position).
• the pool of amino-acyl-tRNA molecules contains an aa-tRNA whose anticodon is complementary to an mRNA codon normally signaling initiation or termination of translation, thus suppressing the signal.
• aa-tRNA whose anticodon is complementary to an mRNA codon normally signaling initiation or termination of translation, thus suppressing the signal.
• a DNA sequence of the present invention will still produce the novel haloalkane dehalogenase(s) either because the insertion(s) of the "wrong" amino acid do not cause the enzyme to lack activity or because the DNA sequence contains - at the position(s) where an "incorrect” amino acid would otherwise be inserted - a codon that "anticipates” the change in the translation system so as to allow either the insertion of the "correct” amino acid or the "correct” signaling of the mRNA codon therein.
• a preferred DNA sequence comprises a polynucleotide substantially homologous with the nucleotide sequence of bases 37-912 of Figure 2.
• this polynucleotide is at least about 90% homologous, even more preferably at least about 95% homologous, and yet more preferably at least about 99% homologous therewith.
• the phrase "substantially homologous” expresses the degree of similarity of a subject sequence - i.e. a subject nucleotide sequence (of an oligo- or polynucleotide or DNA strand) or a subject amino acid sequence (of an oligo- or poly-peptide or protein) - to a related, reference nucleotide or amino acid sequence. This phrase is defined as at least about 75% “correspondence" (i.e.
• alignment is said to exist when a minimal number of "null” elements have been inserted in the subject and/or reference sequences so as to maximize the number of existing elements in correspondence between the sequences.
• "Null” elements are not part of the subject and reference sequences; also, the minimal number of "null” elements inserted in the subject sequence may differ from the minimal number inserted in the reference sequence.
• Increased degrees of homology of a given sequence which may be expressed as, e.g., "90% homologous," are likewise defined with reference to their degree of sequence identicality to a reference sequence.
• a reference sequence is considered "related" to a subject sequence where either: 1 ) both nucleotide sequences encode proteins or portions of proteins which may be identified to the same IUB subclass or 2) both amino acid sequences make up proteins or portions of proteins which may be identified to the same IUB subclass, regardless of whether such identification is based on functional properties, sequence homology, or parental origin.
• Parental origin refers to the fact that a given enzyme may initially be grouped within an IUB subclass because of its recognized major or minor function(s), but after the DNA sequence encoding that enzyme accumulates one or more mutation(s), the encoded enzyme may exhibit functional capacities of a different IUB subclass - whether or not the enzyme also retains its original functionality; the "different IUB subclass” may fall within the same or a different IUB main class.
• portions of proteins signifies that bi- and multi-functional enzymes - including fusion proteins - are also contemplated as falling within a given IUB subclass based on the identification to that subclass of one of their functional domains.
• the haloalkane dehalogenase at least parentally belongs to IUB sub-subclass 3.8.1.
• the enzymes of the present invention have been found to possess unexpectedly superior properties to those of the wild-type haloalkane dehalogenase enzyme found in Rhodococcus as, e.g., was utilized in U.S. Patent No. 5,372,944.
• two characteristics of a given enzyme will determine its usefulness in commercial processes: its affinity for product, as well as its affinity for substrate. Where an enzyme's affinity for product molecules is relatively high, it will be extremely sensitive to feedback inhibition by the product.
• a convenient indicator of an enzyme's relative affinity for product is its inhibition constant measured at 90% inhibition ("r (90)"), i.e. the product concentration at which the enzyme retains only 10% of its Vmax, the Vmax being measured when the concentration of product is 0.
• r (90) the inhibition constant measured at 90% inhibition
• the recombinant enzyme of the present invention has a measured K,(90) of 50 mM.
• the recombinant enzyme is much less sensitive to feedback inhibition by product and can therefore operate in the presence of product concentrations that would essentially shut off the wild type enzyme altogether.
• the enzyme of the present invention may be expressed alone, or covalently attached, along its amino and/or carboxy terminus, to one or more polypeptide tail(s).
• Such tails may be encoded by exons separate from the enzyme-encoding exon or by DNA sequences which are part of the enzyme-encoding exon.
• the tail-encoding DNA may be attached or "fused" to the 3' and/or 5' end of the enzyme gene, e.g., either: 1 ) during enzyme gene amplification by including the tail-encoding nucleotide sequence in an oligonucieotide primer or 2) during plasmid construction by ligating the tail-encoding DNA directly into a plasmid which contains the enzyme gene (whether the enzyme gene is inserted into the plasmid before or after insertion of the tail-encoding DNA). Under the influence of the appropriate genetic control elements - i.e.
• polypeptide tails on one or both ends.
• An example of a preferred tail-free enzyme is that having the amino acid sequence of residues 1 -292 of Figure 2.
• examples of some preferred polypeptide tails include poly-histidine sequences, polyacid (e.g., poly-aspartic and/or - glutamic acid) sequences, cellulose binding domains, and the c-myc, S-Tag, and FLAG peptides.
• Antibodies and affinity columns that bind these exemplary tails are commercially available and may be readily used to purify or immobilize the expressed fusion proteins. However, many other tails may be used while retaining a functional dehalogenase enzyme. Whether or not a tail-encoding sequence is included in the expressed gene, the gene must include, in a position outside the enzyme gene or the enzyme-tail fusion gene, a translation start site, preferably ATG, and will also preferably include an endonuclease restriction site.
• the open reading frame of a single exon encodes a functional dehalogenase enzyme having tails of up to about 30 amino acid residues on the amino and/or carboxy termini.
• the tails may be of approximately equal length.
• the enzyme is expressed with both an amino and a carboxy terminal tail, but the carboxy terminal tail is significantly longer than the amino terminal one.
• the amino- terminal tail is up to about 25 amino acids in length and the carboxy-terminal tail is about 2 to about 150 amino acids in length.
• the amino- and/or carboxy-terminal tail will contain a stretch of at least 5 adjacent histidine residues.
• the amino terminal tail is about 10-150 amino acids in length and preferably contains or is itself a poly-histidine sequence.
• the enzyme may be reversibly immobilized or reversibly inactivated by contact with a surface coated with chelated divalent metal ions, e.g., Mg 2+ or Ni 2+ .
• the poly-histidine- containing amino-terminal tail may be so long as to partially or totally block access to the enzyme's active site.
• the tail may be designed to contain one or more amino acid residues which change the configuration of the tail from that found in a poly-histidine sequence to a bent, recurved, or flexible-joint configuration allowing increased access to the active site of the enzyme.
• the open reading frame encodes a functional dehalogenase enzyme with an amino terminal tail of about 1 to about 25 amino acids and a carboxy-terminal extension having a polyhistidine sequence, a FLAG peptide sequence (available from KODAK Imaging Systems/VWR, Rochester, NY) and/or an S-Tag peptide sequence.
• the open reading frame encodes a functional dehalogenase enzyme having: 1) an amino-terminal tail of up to about 10 amino acids and a polyhistidine sequence and 2) a carboxy-terminal tail comprising (i.e. containing) the FLAG (see Figure 2) or S-Tag peptide sequence.
• the enzymes and/or tails of the above-described dehalogenase enzymes may be modified by use of the techniques of directed evolution, in order to improve their productivity, stability, and/or inhibition profiles.
• One directed evolution technique uses the gene shuffling method disclosed in U.S. Patent No. 5,605,793 to Stemmer et al., in which a number of similar DNA sequences are fragmented and reassembled in a random fashion to generate highly diverse libraries which can be screened for enzymes with the attributes of interest.
• Another version of this technology involves use of error-prone gene amplification technologies.
• a third version of directed evolution employs a combination of these two methodologies.
• a fourth version of directed evolution is the so-called "staggered extension" process as disclosed in the publication by Zhao etal., in Nature Biotechnology (1998) (currently in press).
• error-prone gene amplification is used to introduce semi-random mutations into the dehalogenase gene (e.g., Figure 2, residues 1- 292) at a rate of about 1-6 point mutations per gene copy per gene amplification reaction, following which the mutant library is introduced into bacteria, induced to express protein, and screened for activity, preferably in a spatially addressable grid format (such as a 96 well or a 384 well plate).
• primer-dependent mutagenesis methods e.g., error- prone gene amplification and defined primer-based recombination
• specific protein subdomains can be easily targeted for mutagenesis by primer design and positioning.
• primers are used which allow mutagenesis of the entire transcription and translation domain as it occurs within the expression construct.
• primers are directed exclusively to the protein coding region of the expression construct or target DNA (including tails).
• primers are designed in such a way as to target mutagenesis to the dehalogenase enzyme gene while preserving the sequence of the tails.
• the dehalogenase enzyme gene may be the sole mutagenesis target when an error-prone gene amplification technique employs both a primer complementary to nucleotides closely preceding nucleotide 36 and a primer complementary to nucleotides closely following nucleotide 912.
• the entire Figure 2 coding region is the mutagenesis target when the primers anneal outside of the region of nucleotides 1 -951 ; the Figure 2 amino tail or carboxy tail, respectively, is targeted when the primers anneal outside of the region of nucleotides 1 -36 or 913-951.
• the DNA sequence(s) encoding the enzyme or fusion protein of the present invention will preferably be inserted into an expression vector, followed by transfection of the vector into a host cell, and growth of the host cell under conditions in which it expresses the enzyme.
• a wide variety of recombinant host-vector expression systems for prokaryotic cells are known and may be used in the invention.
• prokaryotes such as those of the genus Bacillus, Pseudomonas, Actinomyces, Bacillus, or Rhodococcus
• eukaryotic microorganisms such as yeast and fungi, e.g., those of the genus Pichia, Saccharomyces, or Aspergillus, e.g., Pichia pastoris or Saccharomyces cerevisiae
• other eukaryotic cells and cell lines such as Sf21 cells infected with baculouvirus-derived vectors
• algal cells are capable of producing, in active form, heterologous proteins of prokaryotic origin; in the event these other cells are utilized in the present invention, appropriate expression vectors would be selected for use therewith.
• prokaryotic expression vectors are available publicly and may be used in the present invention
• expression of the novel enzymes is exemplified herein with the use of commercially available vectors from the pTrcHis, pRSET and pTrxFus series (available from Invitrogen of San Diego, CA, USA) in conjunction with E. coli host cells.
• the DNA of the mutant gene pool produced thereby is digested with appropriate restriction enzymes (i.e. those endonucleases having restriction sites located external to the mutagenesis target); next, the mutant genes are purified and ligated into prokaryotic expression vectors to form a plasmid library.
• Competent host cells e.g., preferably E coli cells, are then transformed with the plasmid library and grown in a suitable medium: in the case of E coli, the cells are plated on agar containing a selective growth medium.
• the cells may then be diluted to form individual clones, or in the case of prokaryotes such as E. coli, they may undergo an initial growth phase, after which the cell colonies are picked individually and transferred to separate containers, e.g., the wells of a 96 well plate, such that each well contains an individual clone of transformed cells.
• individual clones can be expanded, induced to express the protein of interest, and screened for the activity of interest. Screening for the haloaliphatic dehalogenase activity of the novel enzymes is preferably accomplished by detecting the protons or the halide ions released upon hydrolysis of a carbon-halogen covalent bond in a substrate molecule.
• the pH change accompanying the proton release serves as a measure of enzyme activity; this pH change is preferably determined using a fluorescent or visible pH indicator which undergoes measurable color change over the functional pH range of the target enzyme.
• a fluorescent or visible pH indicator which undergoes measurable color change over the functional pH range of the target enzyme.
• multiple parallel pH probes may be utilized.
• the assayed mixture will contain: 1 ) whole cells, permeablized cells, cell lysate, or purified enzymes obtained from cells expressing a mutant dehalogenase, preferably from bacterial cells; 2) a substrate; and 3) a low concentration of buffer (typically ⁇ 10 mM).
• a chemical detergent e.g., sodium deoxycholate
• a physical freeze/thaw process may be used to make bacterial cells permeable.
• the substrate will preferably comprise one or more halogenated aliphatic hydrocarbons as discussed above.
• the buffer may be selected from any known to be effective or found to be effective over the pH range in which the enzyme retains activity.
• the cell debris itself will be seen to provide sufficient buffering capacity to allow accurate quantitation of activity.
• an added buffer it will preferably have a pKa in the range of about 6 to about 10, although other buffers may be used.
• preferred buffers include glycine, 2-AMP, CAPSO, ethanolamine, CHES, borate, serine, and AMPSO; especially preferred is CAPSO and even more preferred is a concentration of about 5mM CAPSO.
• the activity screening assay will also require the use of a detection method.
• a pH change is detected.
• a pH indicator will be included in the assayed mixture. Any pH indicator having a color change in the pH range in which the enzyme is active may be used.
• the pH indicator will undergo a color change in the range of about pH6 to about pH10, more preferably in the range of about pH7 to about pH9.
• Examples of preferred visible pH indicators include m-cresol purple, cresol red, phenol red, bromthymol blue, and thymol blue; examples of preferred fluorescent pH indicators include ⁇ -naphthol sulfonic acid, 1 ,4-naphthol sulfonic acid, coumaric acid, 3,6-dioxyphthalic dinitrile, and orcinaurine.
• a pH probe may be utilized to detect the pH change.
• the visible pH indicator m-cresol purple, and even more preferred is a concentration of about 50 ⁇ M m-cresol purple.
• detection is accomplished by measuring the release of halide ions from the substrate by: 1) including in the assayed mixture a halide- sensitive fluorescent dye, such as lucigenin (available from Molecular Probes of Eugene, OR, USA) - lucigenin is quenched upon contact with halide ions and so a decrease in fluorescence in measured therefrom; or 2) utilizing a halide ion responsive probe device, such as a halide-selective electrode.
• a halide- sensitive fluorescent dye such as lucigenin (available from Molecular Probes of Eugene, OR, USA) - lucigenin is quenched upon contact with halide ions and so a decrease in fluorescence in measured therefrom
• a halide ion responsive probe device such as a halide-selective electrode.
• a coupled enzyme system may be used to detect the production of product molecules: dehalogenation of haloalkanes results in generation of alcohols, and many alcohols are substrates for one or more commercially available alcohol dehydrogenase enzymes (whose activity is measured by disappearance of NADH). Detection of alcohols via coupling to the NADH requirement of the dehydrogenases is well known in the art.
• the enzymes of the present invention may be immobilized onto one or more solid support(s).
• Enzyme immobilization technologies are most conveniently classified into covalent and non-covalent methods.
• Covalent methods utilize reactive groups present on certain amino acid side-chains to bond to a polymeric or inorganic support either directly or by using a bifunctional cross-linking agent. The primary advantage of this approach is the robustness of the linkage.
• Non-covalent immobilization methods are more numerous and range from direct and indirect (e.g., chelate- or chelant-mediated) ionic, adsorptive, or bioaffinity support associations (e.g., biotin-avidin) to gel-entrapment or microencapsulation.
• a preferred method of immobilization involves covalently linking the enzyme to the support by means of reactive groups such as epoxides, activated nucleophiles, isourea, and so forth. These reactive groups may be present on the native surface of the support material or the support material may be modified to bear linkers containing such groups.
• Preferred linkers include those comprising at least one of: dialdehyde, diacid, diamino, diisocyanate, cyanate, and diimide groups; linkers comprising at least one carbodiimide group may also be used, provided that a diamino group is not used in conjunction with a carbodiimide.
• the preferred solid supports are alumina-based supports and silica-based supports; more preferred are polyethyleneimine-impregnated alumina- or silica-based supports.
• a preferred method of immobilization comprises pre-treating the solid support with glutaraldehyde and then contacting the support with the enzyme.
• the enzyme may be conveniently used to convert its substrate/reactant into product.
• This conversion can be performed in any suitable medium which does not substantially affect the activity of the dehalogenase.
• the enzymatic conversion is done in a aqueous medium containing either a buffering system or one or more pH-control devices.
• the halogenated hydrocarbon substrate is generally added to a reaction medium to the saturation point of the substrate, though in some cases, supersaturated substrate mixtures, substrate emulsions, or pure substrate preparations may also be used.
• the concentration of halogenated hydrocarbon used will generally range from about 0.005% to about 0.5% (w/v).
• the concentration of the halogenated hydrocarbon is from about 0.005% to about 0.25%. More preferred is a concentration of halogenated hydrocarbon from about 0.005% to about 0.2% in medium.
• the substrate may be added to the reaction solution initially, as in a batch method, or be added into the liquid stream of a continuous feed process.
• the liquid stream may initially contain substrate or the substrate may be first added thereto as the stream is en route to the reactor. In either case, more substrate may be added directly to the liquid stream in the reactor in order to ensure that a high concentration of substrate is presented to the enzyme throughout the reactor.
• the liquid stream may be re-saturated with substrate at various intervals in the process in order to enable accumulation of product at concentrations higher than the solubility limits of the substrate.
• the batch method reaction is usually carried out with shaking or stirring.
• the reaction time or reactor residence time may vary depending on the reaction conditions, such as the substrate concentration or the amount of enzyme, the reaction conditions are preferably selected so that the reaction is completed within a maximum of 120 hours.
• 1 ,4-Dichlorobutane, 60% perchloric acid, ferric nitrate, and mercuric thiocyanate were from Aldrich.
• Anhydrous ethanol was from Quantum/USI (Tuscola, IL, USA).
• 1 ,2,3- Trichloropropane was a gift from The Dow Chemical Company's Allylics Group (Freeport, TX, USA).
• Monobasic potassium phosphate, dibasic potassium phosphate, imidazole, guanidine hydrochloride, disodium EDTA, ammonium sulfate, and Tris free base were Fisher Biotech Grade.
• Sulfuric acid was from Fisher (ACS grade).
• the Tresyl-Toyopearl chromatography support was from TosoHaas (Lot # 65TRM72R). Sephadex G-25 prepackaged columns were from Pharmacia. Celite R-648 was from Manville. Polyethyleneimine, 50,000 MW and PEI-silica were from Sigma. Glutaraldehyde, Grade 1 , as 25% aqueous solution, also from Sigma, was stored at -20°C until just prior to use. Other samples used in immobilization include: Davison Low SA Alumina, Norton SA 6176 Alumina, Calcicat Type C Alumina, Calcicat s-88-473 Type A Silica, Shell 5980-F Silica, Davison 952-08-5X Silica, Borecker subunit Carbon, and
• DNA Amplification was performed using standard polymerase chain reaction buffers supplied by Perkin-Elmer-Cetus (Nutley, NJ). Typically, 50 ⁇ L reactions include 1x concentration of manufacturer supplied buffer, 1.5 mM MgCI 2 , 125 ⁇ M dATP, 125 ⁇ M dCTP, 125 ⁇ M dGTP, 125 ⁇ M dTTP, 0.1-1.0 ⁇ M forward and reverse primers, 5U AmpliTaq DNA Polymerase and ⁇ 1 ng target DNA. Unless otherwise indicated, thermal profile for amplification of DNA is for 35 cycles of a thermal profile of 0.5 min. @94°C; 1 min. @55°C; 1 min. @72°C.
• Soluble protein was mixed 1 :1 with solubilization buffer (Tris/SDS/ ⁇ -mercaptoethanol, pH 6.8; ISS) and boiled for five minutes before being loaded on 10-20% gels (Daiichi, Natick, MA) and electrophoresed with Tris-glycine buffer (ISS). Gels were stained with Pro-BlueTM (ISS).
• solubilization buffer Tris/SDS/ ⁇ -mercaptoethanol, pH 6.8; ISS
• an RDhl enzyme gene or RDhl fusion protein gene was provided as an EPPCR mutagenesis target, e.g., by using appropriate restriction enzymes to digest a plasmid containing the target DNA sequence.
• the target DNA was purified by gel electrophoresis, followed by gel extraction of the target DNA.
• EPPCR involved performing a standard PCR gene amplification of the target gene, using appropriate oligonucieotide primers, except that the standard PCR buffer was supplemented with sufficient magnesium chloride and manganese chloride to bring the reaction mixture to 7 mM magnesium chloride and 0.15 mM manganese chloride. This procedure may be repeated upon one or more of the EPPCR products to introduce further mutations therein.
• the resulting EPPCR products were ligated into expression vectors (e.g., pTrcHis, pTrxFus) and the vectors were then used to transform appropriate, competent host cells, e.g., E. coli AG1 or JM109 cells, for enzyme expression and enzyme activity analysis. Plasmid-containing clones were identified by selective growth on LB/Amp agar plates. Individual colonies were transferred by toothpick into the wells of a 96-well plate containing a selective growth medium and incubated at 37°C for -8-12hr to allow for growth. Following the initial growth phase, replica plates were generated, expanded, and individual clones thereof were assayed for dehalogenase activity as described in the following section.
• expression vectors e.g., pTrcHis, pTrxFus
• RDhl Enzyme Activity was measured by detecting the pH change resulting from action of the enzyme in dehaiogenating substrate.
• Prokaryotic host cells expressing the enzyme were grown in broth, quantitated, and permeablized prior to addition of a pH indicator, buffer, and substrate.
• Each well of a 96-well microplate received 200 ⁇ L of an SOB broth (obtained from Difco, Detroit, Ml, USA) which had been supplemented with about 50-100 ⁇ g/mL of ampicillin ("SOB/Amp"). Cells from a single colony of enzyme-producing E. coli clone were inoculated into one well of the plate.
• SOB/Amp ampicillin
• each well was inoculated with cells from a different E. coli clone.
• Six wells received no cells, in order to serve as a negative control, and six additional wells were inoculated with an E. coli clone producing the wild-type RDhl enzyme, as a positive control.
• the inoculates were incubated overnight in a Psycrotherm oven at 37°C while being shaken at 250 rpm.
• the cultures were induced by addition of IPTG to a final concentration of 1 mM, followed by another 5 hours of incubation at 37°C in a Psycrotherm oven with 150 rpm shaking. After the 5 hour incubation, the cell density of each culture was determined by use of a 1.573 Vmax/Kinetic Microplate Reader (Molecular Devices,
• Rhodococcus species ATCC 55388 was cultured as described in U.S. Patent No.
• CnBr cyanogen bromide
• Table 1 Sequences of N-terminal and Proteolytic Fragments Derived from Purified Rhodococcus Dehalogenase.
• RDhl 5.4 and RDhl 3.12 were designed to allow amplification and cloning of the open reading frame encoding the Rhodococcus dehalogenase (RDhl) gene in expression system pEXPROK.
• the sequence of RDhl 5.4 was derived from the N-terminal sequence of the protein whereas RDhl 3.12 was designed based on the actual DNA sequence.
• Primers RDhl 5.7 and RDhl 3.13 were designed to generate an RDhl gene in expression system pRSET and pTrcHis
• Primers Trx2++ and Trx- were designed to generate an RDhl gene in expression system pTrxFus.
• Rhodococcus dehalogenase gene was accomplished by amplification from a genomic DNA library as follows. Genomic DNA was isolated from the Rhodococcus ATCC strain 5538 using the methods of PJ. Astunas and K. Timmis (J. Bacteriology 175:4631-4640 (1993)). Purified genomic DNA (100 ⁇ g) was sheared mechanically to an average size of ⁇ 10 kbp. Fragments were ligated to BamH I linkers, followed by SamH I digestion and gation into a SamH I digested preparation of bacteriophage Lambda-ZAP ExpressTM DNA (obtained from Stratagene, Inc. of LaJolla, CA, USA).
• a library containing the genomic Rhodococcus DNA fragments was prepared commercially (Stratagene, Inc., LaJolla, CA) and supplied at a titer of 1x10" pfu/ ⁇ L (plaque forming units per microhter).
• a redundant DNA primer (RDhl 5.4) corresponding to the codons for amino acids 6-13 of the N-terminal sequence was synthesized using solid phase phosphoramidite chemistry and purified by HPLC (Table 2).
• the RDhl 5.4 primer was used in combination with a commercially available primer which recognizes the T3 bacteriophage promoter sequence (and is contained within the Lambda ZAP ExpressTM vector) to amplify dehalogenase sequences from the singly- expanded genomic DNA bacteriophage library.
• Amplification was accomplished using the polymerase chain reaction (50 ⁇ L) containing 1 ⁇ M of RDhl 5.4 primer, 100nM biotinylated T3 Pro primer (New England Biolabs), 10x Amplitaq reaction buffer (Perkin-Elmer-Cetus), 1.5 mM MgCI 2 , 5U of rAmpliTaq DNA polymerase (Perkin-Elmer-Cetus), and 4 ⁇ L of the phage library (whole phage). Amplification was for 35 cycles of the following thermal profile: 1 min. @94°C; 2 min. @ 55°C; 2 min. @72°C.
• PCR products were separated by electrophoresis through 1.0% agarose and a discrete band of 1.3 kbp was identified, excised from the gel, and isolated using a QiaQuick gel purification kit (Qiagen, Inc.). After confirming that this DNA was also capable of being amplified by other Rhodococcus dehalogenase-specific primers, the fragment was digested with restriction enzymes Nco I and Pst ⁇ and ligated into Nco ⁇ /Pst ⁇ digested pGEM5zf(+) (ProMega, Madison, WI). Sequencing of the 3'-untranslated region of the cloned segment allowed identification of a putative stop codon and subsequent amplification of the coding region with primers RDhl 5.4 and RDhl 3.12.
• Double stranded sequencing of the dehalogenase gene proceeded via successive rounds of the dideoxy method with the biotinylated primers (Table 3) designed for each successive round, based on the sequencing results in preceding rounds. Bands were separated on 5.5-6.0% polyacrylamide urea sequencing gels, the DNA transferred to nitrocellulose filters by capillary transfer and visualized using the well-known streptavidin-alkaline phosphatase development protocols in combination with chemiluminescent substrates.
• Table 3 Sequences and Orientation of Oligonucieotide Primers Used in Sequencing the Rhodococcus Dehalogenase Gene
• the vector pEXPROK ( Figure 1 ) is a derivative of the commercially available pPROK-1 vector (Clontech, Inc., Mountain View, CA). Whereas the pEXPROK retains the functional elements of the pPROK vector (including the ampicillin resistance marker, the Ptac transcriptional promoter, and paired transcription termination signals following the polylinker), pEXPROK vector replaces the EcoR l-to- Hind III polylinker of pPROK-1 with an extended synthetic polylinker referred to as EXFLAG.
• the EXFLAG linker is designed to allow insertion of an open reading frame between an Nco I site and an Nhe I site.
• This procedure was used to insert the RDhl gene into pEXPROK.
• the plasmid maps of pEXPROK and pEXPROK-RDhl are shown in Figures 1 and 3, respectively. The DNA sequence of the pEXPROK-RDhl construct was later confirmed by automated DNA sequencing.
• DNA Sequence Analysis The complete DNA and derived protein sequences for the dehalogenase gene are shown in Figure 2.
• DNA Sequence data reveals an open reading frame of 876 bp, giving a deduced protein sequence of 292 amino acids and a predicted molecular weight of 33kD. This is similar to the molecular weight reported for a number of other hydrolytic dehalogenases.
• a MacVector v.4.5.2 Kerat, Inc. sequence analysis package was used to compare the derived protein sequence with those of all other known proteins contained in the Entrez Sequence Database (the Entrez Database is maintained by the National Center for Biotechnology Information).
• the RDhl polypeptide displays the greatest similarity to members of the so-called ⁇ / ⁇ hydrolase family of enzymes including several haloalkane and haloacid dehalogenases, epoxide hydroalses, and enzymes with a number of diverse catalytic functions.
• cloned enzyme as a dehalogenase we sought to express the full-length protein in E. coli.
• a 1300 bp Nco USpe I restriction fragment, containing the RDhl gene was excised from the pRDhlK02.1-pGEM5 construct and ligated with the Nco ⁇ INhe l-digested pEXPROK vector.
• Colonies transformed with the resulting plasmid (pRDhlK02.3-EXPROK) were grown overnight in 2 mL minicultures, following which 1 mL of each culture was pelleted, washed, and sonicated. Extracts were then assayed for their capacity to catalyze release of chloride following addition of the RDhl substrate, 1 -chlorobutane. Chloride releasing activity was absent from cultures not containing the cloned gene; cultures with the cloned gene exhibited chloride releasing activity which increased when transcriptional activity of the gene was increased by the addition of IPTG. Thus, dehalogenase activity could be induced in overnight cultures of the recombinant E. coli containing the pRDhlK02.3-pEXPROK construct.
• the RDhl open reading frame was amplified with primer RDHL 5.4 as the forward primer (containing an Nco I site to direct the start of translation) and primer RDHL 3.12 as the reverse primer (and containing an Nhe I site).
• primer RDHL 5.4 as the forward primer (containing an Nco I site to direct the start of translation)
• primer RDHL 3.12 as the reverse primer (and containing an Nhe I site).
• the gene was ligated into the pEXPROK vector.
• the new construct was then transformed into E coli AG1 competent cells and ampicillin resistant colonies were picked. Plasmids containing the RDhl gene were identified by analytical restriction enzyme digestion and referred to as pEXPROK-RDhl construct.
• the pEXPROK-RDhl plasmid map is shown in Figure 3. Construction of pRSET-RDhl and pTrcHis-RDhl Expression Vectors
• both pRSET-RDhl and pTrcHis-RDhl expression vectors were amplified from the pEXPROK-RDhl using oligonucieotide primers RDhl 5.4 and RDhl 3.13 using standard PCR conditions. Amplification products were separated on agarose gels and purified using standard procedures.
• Both pRSET and pTrcHis vectors are IPTG inducible expression vectors, derived from the pUC 18 and 19 series of cloning vectors. They both were purchased from Invitrogen Corp. (San Diego, CA) and contain the following features:
• Both are designed for high level protein expression and both carry an ampicillin resistance gene.
• Both contain a sequence that encodes an N-terminal fusion peptide which codes for six histidine residues. These residues function as a metal binding domain and may allow later purification of recombinant protein by affinity chromatography.
• the vectors encode an enterokinase cleavage recognition sequence (the FLAG and/or EXFLAG peptide) downstream of the dehalogenase coding region which allows detection by and immobilization upon anti-FLAG antibodies.
• the high level expression property of the pRSET vector results from the presence of the T7 promoter upstream of the heterologous gene. Since E. coli does not contain the T7 polymerase, however, an M13 phage containing the T7 RNA polymerase gene is needed for protein expression. In practice, bacteria containing a heterologous gene under the control of a T7 promoter are induced to produce the heterologous protein by infection of recombinant E. coli with T7 phage containing the T7 RNA polymerase. Alternatively, commercially available E. coli stably expressing the T7 RNA polymerase enzyme (i.e. BL21 ) can be transformed with the pRSET construct.
• the pRSET-RDhl expression vector was generated by digesting plasmid pRSET with restriction enzymes Nco ⁇ IHind III and then incorporating an RDhl gene fragment which contains an Nco I site at the 5 ' end and a Hind III site at the 3 ' end. The new construct was then transfected into E. coli JM109 competent cells and ampicillin resistant colonies were picked. Plasmids containing the RDhl gene were identified by analytical restriction enzyme digestion and referred to as the pRSET-RDhl construct. The pRSET-RDhl expression construct is shown in Figure 6. One such clone (Clone 16-4) was used to characterize protein expression using the pRSET system.
• the pTrcHis vector contains another high level transcriptional promoter - the trc promoter, a fusion of the well-characterized trp promoter and the lac promoter.
• the pTrcHis vector also contains a mini-cistron upstream of the heterologous gene which provides highly efficient, repeat initiation of translation of the cloned protein in the multiple cloning site.
• FIG. 7 shows a map of the completed pTrcHis-RDhl expression construct.
• One such clone (Clone 18-3) was identified as a high expressing clone and used for further characterization of the TrcHis expression system.
• the ThioFusionTM expression system provides a means of expressing large amounts of heterologous protein by fusing the gene encoding such a protein to the gene encoding the E. coli protein, thioredoxin, in the pTrxFus expression vector.
• the thioredoxin moiety can confer both solubility and heat stability to its fusion partner, thereby opening up new options for purification by osmotic shock or heat treatment.
• the expression vector, pTrxFus allows foreign genes to be inserted into its multiple cloning site.
• P L promoter from bacteriophage lambda to drive expression and the cl repressor, also from bacteriophage lambda, to control the level of transcription.
• Expression of the cl repressor gene is under control of the trp promoter and repressor. Expression of a foreign gene is induced by adding tryptophan to the medium which shuts down cl repressor synthesis and allows transcription from the P L promoter.
• Trx2++ and Trx- were designed to modify the RDhl gene fragment with an enzyme restriction site unique to the TrxFus multiple cloning site.
• Plasmid pEXPROK-RDhl was used as a template, and a gene fragment was generated by PCR, using primers Trx2++ and Trx-, which added a BamH I site to the 5 ' end and a Pst I site to the 3 ' end. The fragment was purified using a QIAquick PCR Purification Kit. Both the pTrxFus vector and the gene fragment were enzyme-digested, agarose gel purified, and ligated. The new construct, pTrxFus-RDhl ( Figure 8), was incorporated into GI174 electrocompetent cells (Invitrogen Corp.) which had been prepared following the manufacturer's instructions.
• clones identified as containing proper DNA constructs were cultured in 3 mL of Luria Broth (LB) or SOB medium (Difco, Detroit, Ml, USA) containing 50 ⁇ g/mL ampicillin in 15 mL round-bottom polypropylene culture tubes. These culture tubes were incubated overnight at 37°C with shaking (200 cycles/minute in a rotary shaker) or grown to an OD 600 of 0.6. Afterward, 2 mL of fresh medium with IPTG was added (to a final IPTG concentration of 1 mM) and the tubes were incubated at 37°C with constant shaking for another 4-5 hours. For recombinant clones of pRSET, after 1 hour of IPTG induction the cell cultures were infected with previously titered M13/T7 phage and the incubation continued as described previously.
• LB Luria Broth
• SOB medium Difco, Detroit, Ml, USA
• RDhl gene-containing clones were cultured in 1 mL RM medium with 100 ⁇ g/mL ampicillin and incubated overnight at 30°C with shaking (200 cycles/minute in a rotary shaker). The next day, 9 mL fresh Induction Medium were added and growth continued at 30°C to an OD 550 of 0.5. Then, cell cultures were induced with tryptophan (to a final concentration of 100 ⁇ g/mL) and transferred to a 37°C incubator and shaken at 200 rpm for another 2 to 4 hours.
• Insoluble debris was removed by centrifugation at 10,000 rpm for 10 minutes. Cell-free supematants were then transferred to clean polypropylene tubes and appropriate assays performed. Final cell suspensions from clones of pTrxFus were sonicated for three 10-second bursts and then flash-frozen in a dry ice/ethanol bath. Shortly after freezing, the cell lysates were quickly thawed at 37°C and two more, rapid sonication-freeze-thaw cycles were performed. After the last thaw, the procedures described above for removing the cellular and insoluble debris were continued.
• FIG. 9 shows a Pro-BlueTM stained SDS-PAGE gel of cell lysate samples of the pEXPROK-RDhl clone 12-4 on the left side (lanes 2-5) and partially purified rRDhl enzyme on the right side (lanes 8-11 ).
• Lane 1 contains molecular weight standards and lanes 6 and 7 contain single, 60 ng and 180 ng bands of the FLAG-peptide protein at a molecular weight of 55kD.
• Lanes 2-5 show all the soluble protein from cell-free extracts. Since rRDhl enzyme is not a major protein in the extracts, immunoblotting of an identical gel was done to confirm the presence of this recombinant enzyme.
• Figure 10 shows this recombinant enzyme band in each sample lane, as recognized by an anti-FLAG antibody at the predicted molecular weight of - 35kD.
• Lanes 6 and 7 in Figure 10 are 20 ng and 60 ng, respectively, of the FLAG-peptide protein.
• Affinity purified recombinant enzyme was analyzed on both a Pro- BlueTM-stained SDS-PAGE gel and an immunoblotting membrane.
• Four consecutive fractions of affinity-purified rRDhl enzyme were run in lanes 8-11 of the Pro-BlueTM-stained SDS-PAGE gel shown in Figure 10. In addition to a prominent band at ⁇ 35kD molecular weight, other protein bands are visible on the gel.
• Lanes 2-6 show samples of 1 ⁇ L of cell-free extracts from the 5 clones and lanes 9-12 show samples of 0.1 ⁇ L of the cell-free extracts. Immunoblots of these extracts reveal doublet bands (35kD and 38kD) in each sample lane, when the anti- FLAG antibody is used to stain the immunoblots ( Figure 12). This may suggest that there are two start codons in the pRSET-RDhl system. The first start codon was originally designed in the pRSET vector system to be about 41 amino acids (123 bp) before the actual cloning site, which allows the initiation of translation from that Met ATG codon followed by 6 histidines.
• the second start codon may occur at the Nco I cloning site itself, which was designed into the original 5 ' end primer of the RDhl gene fragment. However, the presence of an anti-FLAG antibody-reactive band confirms the presence of rRDhl enzyme.
• Figure 13 shows the Pro-BlueTM-stained SDS-PAGE gel with cell-free extracts from the pTrcHis system and Figure 14 shows the immunoblot of an identical SDS-PAGE gel.
• All clones of the pTrcHis system show an overloaded, anti-FLAG-reacted band at a molecular weight of -35 kD, which confirmed the presence of rRDhl enzyme in the extracts. Since the volumes of the initial culture and the cell-free extract preparations are the same in all three systems, these overloaded bands are an indication of higher enzyme production in the pTrcHis system.
• FIG. 15 shows a gel stained with Pro-BlueTM.
• Lane 12 shows molecular weight standards and lane 11 shows a single 150ng band of the FLAG-peptide protein at a molecular weight of 55kD.
• the thioredoxin fusion bands are clearly visible as the major protein in lanes 1 to 9 at a molecular weight of 47 kD. This size corresponds to the 12 kD of the thioredoxin protein and 35 kD of the rRDhl enzyme.
• lane 10 has a sample of insoluble matter from cell lysis, which shows no presence of the high-level, expressed thioredoxin fusion protein. This demonstrates that all of the fusion protein is in a soluble state. Data from other experiments indicate this fusion protein band can be recognized by anti-FLAG antibody at the same molecular weight in the immunoblot membrane (data not shown).
• Recombinant protein activity was measured by adding an appropriate amount of cell- free extract (prepared as described above) to 6.0 mL of reaction buffer in a glass vial.
• 100 mM sodium glycinate buffer (pH 9.0) was used for measuring activity toward 1 ,4- dichlorobutane (DCB) (Aldrich Chemical Co.), and 100 mM Tris-S0 4 buffer (pH 7.0) was used for measuring activity toward 1 ,2,3-trichloropropane (TCP).
• the halogenated substrate (6 ⁇ L) and a micro stir bar were added and the vial was capped. Capped vials were incubated in a 30°C water bath with stirring.
• Rhodococcus haloalkane dehalogenase can be expressed at high levels in E Coli in 3 of the 4 systems examined.
• the recombinant Rhodococcus dehalogenase is stably expressed in all four systems and recognized by anti-FLAG antibodies at the expected molecular weight.
• This recombinant enzyme exhibits a dehalogenase activity at a level similar to that of the wild type and proportional to the level of heterologous protein expression.
• Porous Alumina Supports Protein (385 mg) representing 22 TCP units of activity were immobilized on 2.0 g of volatile-free alumina (lot #1587 of k-4 alumina from UOP of DesPlaines, IL, USA) at 4°C with mild agitation over the weekend. The procedure followed UOP's standard practice of GIA- activation of the polyethyleneimine coating, water washing, and enzyme addition. The bathing solution was decanted and the support was washed five times with 2 mM Tris/1 mM EDTA, pH 7.5.
• Rhodococcus dehalogenase was produced in E. coli using the pTrcHis expression system. Enzyme preparations used for all immobilization studies were first partially purified using ammonium sulfate precipitation, using a cut of 45 to 70% saturation at 4 °C, followed by dialysis and clarification in 10 mM Tris sulfate, 1 mM EDTA, pH 7.5. This basic buffer was used throughout all purification steps. These preparations were routinely 4.5-fold purified from the lysate, as determined by absorbance at 280 nm, and were estimated to be 30-35% pure dehalogenase protein by SDS-PAGE.
• More highly purified enzyme preparations were achieved by an additional DEAE-Sepharose chromatographic step of eluting with a 0-400 mM ammonium sulfate gradient. This provided about 10-fold purification from lysate, with 85-90% enzyme purity. This step was followed by QAE- Sepharose FF chromatography with a narrower 0-120 mM ammonium sulfate gradient, achieving about 12-fold purification from lysate, and SDS-PAGE which demonstrated enzyme homogeneity.
• Purified RDhl from the TrcHis RDhl expression system is typically referred to herein as "rRDhl.”
• Inorganic supports were modified with polyethyleneimine and glutaraldehyde according to well established protocols (U.S. Patent No. 4,268,410 and Mosbach,
• the rRDhl preparation used for these studies had been affinity purified from E coli lysate using the anti-FLAG antibody column, and was estimated to be about 20% pure by SDS-PAGE. 0.35 units of enzyme (1.96 mg total protein) were coupled to 40 mg of the Tresyl-Toyopearl under conditions described by the manufacturer. After 3 hours, 93% of protein had been coupled as determined by the decrease observed at A 280 . An additional 10 mg of resin were added and coupling continued for 1 hour to bind >98% of the protein. Re- assay of the washed gel for dehalogenase activity revealed recovery of 0.11 units of activity (31 %).
• Inorganic supports have also found wide utility in the industrial enzyme arena due to availability, low cost, high loading capacity, ease of regeneration and reuse, and the wide range of pore sizes. Porous alumina, silica, and Celite have found widespread use as supports for immobilized enzymes, with titanium- and carbon-based supports seeing more limited application.
• Enzymes can be immobilized to inorganic supports by three mechanisms.
• the enzyme may associate with the inorganic support through ionic interactions or may bind through an ion-exchange mechanism to an ionic polymer which has been impregnated into the inorganic support, or be crosslinked to the ionic polymer using a bifunctional chemical linker.
• the first approach has not seen wide applicability because the weak ionic interactions frequently lead to enzyme leaching.
• Polyethyleneimine (PEI) is the polymer of choice for impregnation because of low cost.
• the amino groups allow a wide range of crosslinking chemistry to be applied. Glutaraldehyde is by far the most studied and inexpensive crosslinking agent used. Studies with rRDhl focused entirely on this coupling chemistry.
• Both supports were treated with glutaraldehyde (GIA) and then washed exhaustively with water. 5.5 units (1.0 mL at 0.55 mg protein/mL) of a highly purified rRDhl enzyme preparation (>98% pure by SDS-PAGE) was used to couple to 500 mg each of the two glutaraldehyde- treated, PEI-impregnated supports. This loading level (0.11 % w/w) was assumed to be at least two orders of magnitude below the known loading capacity of the supports. The enzyme was incubated with the supports, with gentle shaking overnight (18 hr) at 4 °C, before washing exhaustively to remove unlinked protein.
• GAA glutaraldehyde
• Re-assay of the two supports with DCB demonstrated recoveries of 40% for the PEI-Celite R-648 and 31% for the PEI-Silica. These samples were stored at room temperature under reaction conditions (saturating DCB) for 1 week and re-assayed.
• the Celite immobilized enzyme preparation lost 49% of activity in a week while the Silica immobilized enzyme preparation lost 28% of its activity.
• Each support exhibits a different uptake profile ranging from 62% to 95% uptake after 72 hours. For most systems, 72 hours is adequate to achieve maximum loading of protein achievable at 4 °C. However, the three alumina systems actually showed greater uptake at 24 hours than at 72 hours. Recovery of bound enzyme activities at 72 hours ranged from 3% to 21 % as compared to an untreated soluble enzyme control. Considerable activity was unaccounted for or "lost" in all systems examined, ranging from 55% to 87%. Also, the previously run supports (Sigma PEI-Silica and Manville Celite R648) showed poorer bound recoveries. This could be due to either the lower enzyme loading ratio or the longer incubation times used in this experiment.
• PEI The molecular weight of PEI is also known to have an impact on the overall yield and stability of immobilized enzymes.
• PEI is capable of functioning either as an ion exchange ligand on various supports or as a glutaraldehyde cross-link acceptor.
• PEIs of two different molecular weights were impregnated onto various porous inorganic supports following the method described in the previous section. In these experiments, however, enzyme (semi-purified preparations containing 1-2 U/mL) was bound by ion-exchange but the GIA crosslinking step was omitted. Samples were submitted for a stability screen to determine if the size of the PEI was an important factor. Table 9 Stability Screen of PEI-impregnated Porous Supports. No Crosslinking
• the pRSET-RDhl.Nde expression vector was generated by digesting plasmid pRSET RDhl clone 16-4 with the restriction enzymes, Nde I and Hind III, and then incorporating into the construct a RDhl gene fragment which contained a Nde I site at its 5' end and a Hind III site at its 3' end.
• the new construct was then transformed into E coli JM109 competent cells (from Invitrogen of Carlsbad, CA, USA) and ampicillin resistant colonies were picked. Plasmids containing the RDhl gene were identified by analytical restriction enzyme digestion and referred to as the pRSET-RDhl.Nde construct. Production of Recombinant RDhl Protein
• DCB unit is a measure of dechlorination activity toward 1 ,4-dichlorobutane (DCB).
• E. coli AG 1 chemically competent cells were purchased from Stratagene (La Jolla, CA, USA);
• E. coli TOP 10F' chemically competent cells were purchased from Invitrogen (Carlsbad, CA, USA); E. coliQ ⁇ 174 cells were purchased from Invitrogen (Carlsbad, CA) and were made electro-competent according to the supplier's instructions.
• the His-6-F and His-6-R oligonucleotides were annealed, ligated into the digested 18-3 construct, and transformed into competent E. coli TOP10 F' cells. Transformed colonies were selected by growth on LB/Amp agar plates. The resulting amino terminal sequence was:
• the carboxy terminus EXFLAG was eliminated by digesting pTrcHis RDhl 18-3 with Avr II and Nhe ⁇ and re-ligating the plasmid. Individual clones were screened by enzymatic digestion and gel electrophoresis.
• Candidate clones were grown at 37° C in 5 mL cultures, induced with IPTG, and lysed by sonication. The lysates were analyzed by PAGE, Western blot, and chloride detection assay. Those clones lacking the amino terminal poly-histidines or the carboxy terminal EXFLAG demonstrated catalytic activity equal to the original construct.
• CTERM S-Tag F forward
• CTERM S-Tag R reverse
• the sequences of these oligonucleotides are as follows (each strand of the S-Tag fragment is underlined): --Avr II—
• the plasmid pTrcHis RDhl-S-Tag was digested with the restriction enzymes Avr W and Not I and ligated with the S-Tag fragment (the S-Tag fragment was prepared by annealing primer CTERM S-Tag F and primer CTERM S-Tag R together at room temperature).
• the new construct, pTrcHis RDhl-S-Tag was incorporated into E coli AG 1 competent cells (from Stratagene of La Jolla, CA, USA) and ampicillin resistant colonies were picked. Plasmids containing the S-Tag fragment were identified by analytical restriction enzyme digestion.
• rRDhl Semi-purified rRDhl (the EXFLAG-tagged protein derived from TrcHis RDhl Clone 18-3) was compared kinetically with semi-purified RDhl-S-Tag protein produced in this example. Chloride-releasing activity was examined at TCP concentrations ranging from OmM to 5mM. As shown in Figure 19, the S-Tag-modified protein exhibited a consistent increase of about 15% in Vmax over the EXFLAG-modified protein. The S-Tag protein also exhibits a -25% lower Km for TCP than does the EXFLAG protein. These results confirm that changes at the C-terminal end of the TDhl enzyme can be used to modulate and improve activity of the enzyme.
• a bench-scale reactor was assembled using all 316 stainless steel with 14 inch ID, in a shell and tube design. Reactor inlet and outlet tubing were also stainless steel. A Lauda circulating water bath (with a thermostat) was used to maintain the reactor at 30°C. The reactor was packed with immobilized rRDhl enzyme running in an up-flow direction. The immobilized rRDhl enzyme was first prepared by loading a partially purified enzyme preparation (approximately 70% purity by SDS-PAGE) onto PEI-impregnated alumina (ISP 4000 grade from UOP) which had been pretreated with 25% (w/v) glutaraldehyde for 2 hours; this was followed by extensive washing with distilled water.
• a partially purified enzyme preparation approximately 70% purity by SDS-PAGE
• PEI-impregnated alumina ISP 4000 grade from UOP
• Sufficient protein was introduced to the alumina to provide 300 mg protein (by Lowry method) per gm of support. Binding was allowed to occur overnight at room temperature. Bound enzyme activity was estimated by measuring unbound enzyme activity in the bathing solution or by final absorbance at 280 nm.
• the immobilized rRDhl enzyme was transferred to the reactor using 2 mm glass beads as spacers at the inlet and outlet. Flow was initiated using an aqueous feed of a pre- warmed 10 mM sodium phosphate/10 ⁇ M EDTA buffer (pH 7.0). After several hours of wash to remove any unbound enzyme, the aqueous feed was saturated with 1 ,2,3- trichloropropane (TCP) and delivered as a continuously stirred solution at a flow rate of 0.15 mL/min. The reactor was allowed to equilibrate for - 2 residence times before sampling the inlet and outlet streams for analysis of reactant TCP and product 2,3-dichloro-1-propanol (DCH) concentrations by GC.
• TCP 1,2,3- trichloropropane
• each sample was first saturated with sodium sulfate and then extracted with chloroform (2 volumes) containing 10mM each of two internal standards (1 ,1 ,1 ,2-tetrachloroethane and 3-chloro-1-propanol). TCP and DCH levels were then estimated from the GC data using the internal standard method and the productivity (the percent yield per volume per time) was calculated therefrom. This initial productivity was used as a measure of initial enzyme activity.
• Error-Prone PCR Mutagenesis was performed upon >4gel- and ⁇ // ⁇ el-digests of plasmid pT re/His RDhl 18-3 which had been purified by agarose gel electrophoresis and extracted from the gel.
• the EPPCR products were ligated into expression vectors having Age ⁇ and Nhe ⁇ cutting sites.
• the resulting plasmids were transformed into competent AGI cells which were grown into colonies on ampicillin-supplemented agar.
• the resulting cell clone "pTric/His RDhl" EPPCR library was tested using the procedure for measuring RDhl enzyme activity by detection of pH change, as follows: wells B1 to H12 of a 96-well microplate, each containing 200 ⁇ L SOB/Amp broth, was inoculated with a single colony from the pT re/His RDhl EPPCR library; wells A1 -A6 contain only media as a negative control, and wells A7-A12 were inoculated with wild type pTrc/His RDhl 18-3 colonies as a positive control. Representative results are presented in Figures 17 and 18.
• NAME The Dow Chemical Company STREET: 1790 Bldg . Washington Street CITY: Midland STATE: MI COUNTRY: U.S.A. POSTAL CODE: 48674 TELEPHONE: 517-636-1687 TELEFAX: 517-638-9786
• ORGANISM Rhodococcus rhodocrous INDIVIDUAL ISOLATE: TDTM003
• NAME/KEY Amino-terminal poly-His tail LOCATION: -10.. -1
• Glu Pro Ala Asn lie Val Ala Leu Val Glu Ala Tyr Met Asn Trp Leu
• SEQ ID NO: 4 Rlucif aa.
• GAATTCAGCC ATGGCATAAG CTTTCTAGAC TCGAGGGAGC TAGCGGCCTA GGTGACTACAA GGACGATGAT GACAAATAAT GAGCGGCCGC TAGCTT

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