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WO1998014595A1 - Lichenase fongique et sequences codantes - Google Patents

Lichenase fongique et sequences codantes Download PDF

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Publication number
WO1998014595A1
WO1998014595A1 PCT/US1997/017811 US9717811W WO9814595A1 WO 1998014595 A1 WO1998014595 A1 WO 1998014595A1 US 9717811 W US9717811 W US 9717811W WO 9814595 A1 WO9814595 A1 WO 9814595A1
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WIPO (PCT)
Prior art keywords
lichenase
sequence
amino acid
nucleotide
seq
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PCT/US1997/017811
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English (en)
Inventor
Xin-Liang Li
Lars G. Ljungdahl
Huizhong Chen
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University Of Georgia Research Foundation Inc.
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Application filed by University Of Georgia Research Foundation Inc. filed Critical University Of Georgia Research Foundation Inc.
Priority to EP97909941A priority Critical patent/EP0931153A1/fr
Priority to AU47435/97A priority patent/AU721534B2/en
Priority to CA002267078A priority patent/CA2267078A1/fr
Publication of WO1998014595A1 publication Critical patent/WO1998014595A1/fr
Priority to US09/286,690 priority patent/US6103511A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01073—Licheninase (3.2.1.73)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2448—Licheninase (3.2.1.73)

Definitions

  • the present invention relates to polysaccharide-degrading enzymes, especially to the enzymes, in particular, to a lichenase enzyme which is capable of degrading (l ,3-l ,4)-/3-glucans and sequences encoding lichenase enzymes.
  • Hemicellulose non-cellulosic polysaccharides including glucans, mannans and xylan
  • the mixed-linked 1 ,3-1 ,4- ⁇ -glucans form the major part of cell walls of cereals like oat and barley.
  • 3-Glucans consist of glucose units jointed by 0-1 ,4 and (3-1,3 linkages, and include lichenan and barley /3-glucan.
  • ⁇ -Glucan accounts for up to 70% of the cell wall in barley endosperm (Guliga and Brant, 1986).
  • Endo-l,3-l,4-j3-D-glucanohydrolase cleaves 3-l ,4 linkages adjacent to (3-1,3 in glucans yielding chiefly cellobiosyltriose and cellotriosyltetraose (Fleming and
  • 0-Glucanase is especially interesting to the brewing industry because 3-glucans cause problems in filtration processes (Godfrey, 1983).
  • /3-glucanase also has application in the poultry industry; it has been added to broiler chick feedstuffs to improve digestibility (White et al., 1983).
  • /3-Glucanases have been cloned from several Bacillus species, including Bacillus subtilis (Murphy et al., 1984), B. amyloliquefaciens (Hofemeister et al., 1986), B. macerans (Borriss et al 1990), B.
  • licheniformis (Lloberas et al., 1991), B. brevis (Louw et al., 1991), B. polymyxa (Gosalbes et al., 1991), and from other genera, including Clostridium thermocellum (Schimming et al., 1992; Zverlov et al., 1992), Fibrobacter succinogenes (Teather and Erfle, 1990), Ruminococcus flavefaciens (Flint et al., 1993), Rhizobium meliloti (Berker et al., 1993, and Cellvibrio mixtus (Sakellaris et al., 1993).
  • a cDNA clone encoding barley /3-glucanase has been isolated and sequenced from germinating barley (Fincher et al., 1986).
  • 1,3-1,4-/3- D-glucanases are known to be produced only by plants and certain bacteria (Borriss et al., 1990; Fincher et al. , 1986). No fungal 1 ,3-1 ,4- ?-glucanases which lack the ability to degrade /3-(l ,4)-glucans are believed to have been discovered prior to the present invention.
  • Obligately anaerobic fungi are part of the natural microflora of the alimentary tract of many herbivorous mammals (Orpin and Joblin, 1988). Since the first strictly anaerobic and filamentous fungus Neocallimastix frontallis was isolated in 1975 from the rumen of a sheep (Orpin, 1975), at least thirteen different anaerobic fungi have been isolated from ruminant and nonruminant herbivores (Chen et al., 1995a). Anaerobic fungi are divided into two groups based on morphology.
  • Neocallimastix Orpin, 1975
  • Caecomyces Caecomyces
  • Piromyces species Barr et al., (1989) Can. J. Botany 67:2815-2824
  • the other is polycentric and it contains Orpinomyces (Barr et al . , ( 1989) supra)
  • Anaeromyces Bostodian et al . , 1990
  • Ruminomyces Ho and Bauchop, 1990.
  • the anaerobic fungi produce a variety of enzymes that degrade plant materials ingested by the host animals (Borneman et al., 1989).
  • a lichenase is an enzyme which hydrolyzes the ⁇ -1 ,4-glucan bonds adjacent to ⁇ -1 ,3-linked glucan bonds, but does not cleave ⁇ -l ,4-linked glucans.
  • Substrates for lichenase include, without limitation, lichenan and barley ⁇ -glucan.
  • the lichenase is selected from the group consisting of that naturally produced by Orpinomyces PC2 (SEQ ID NO:2, amino acids 1 to 216) and that recombinantly produced, for example, in Escherichia coli (SEQ ID NO:2, amino acids -8 to 216).
  • the complete amino acid sequence of the exemplified lichenase, including the signal sequence, is given in SEQ ID NO:2, amino acids -29 to 216.
  • nucleotide sequences which encode a lichenase enzyme of the disclosed specificity and having an amino acid sequence as given in SEQ ID NO:2, amino acid 1 to amino acid 216 or as given in SEQ ID NO:2, from amino acid -8 to amino acid 216 or as given in SEQ ID NO:2 from -29 to 216, for a lichenase with signal sequence. Variations from the specifically exemplified sequence are permitted, to the extent that the functionality of the enzyme is not changed.
  • Orpinomyces PC2 coding sequences for a mature natural lichenase is as given in SEQ ID NO: l, nucleotides 210-860; for the recombinantly expressed lichenase, SEQ ID NO: l, nucleotides 186-860, and for the complete coding sequence including the signal peptide, SEQ ID NO:2, nucleotides 123-860, and sequences with at least about 70% homology to the recited Sequences.
  • Synonymous codings are within the scope of the present invention, and are well within the grasp of the ordinary skilled artisan without the expense of undue experimentation, given the teachings of the present disclosure taken with what is well known to the art.
  • the complete coding sequence (SEQ ID NO: 1 , nucleotides 123-860) is operably linked downstream of promoter sequences appropriate to the recombinant host cell in which expression is desired.
  • the expressed lichenase protein be intracellular, then the coding sequence for lichenase (either as given in SEQ ID NO:l, nucleotides 186-860 or as given in SEQ ID NO: l, nucleotides 210-860) is joined immediately downstream of a translation start signal (ATG) and operably linked downstream of a promoter appropriate to the host cell of choice.
  • ATG translation start signal
  • Recombinant cells which express lichenase are cultured under conditions suitable for the expression of the lichenase coding sequence.
  • an inducible promoter is used to control the expression of the lichenase coding sequence
  • a further object of the present invention is to provide a method for the expression of a lichenase protein of the present invention.
  • This method includes the step of producing a non-naturally occurring recombinant DNA molecule as described hereinabove, with the lichenase coding sequence operably linked to transcriptional and translational control sequences suitable for the host cell of choice, said combination being incorporated within a vector plasmid or virus suitable for the chosen host cell, introducing that recombinant DNA molecule into the host cell to produce a recombinant host cell, and culturing the recombinant host cells under conditions suitable for expression of the lichenase coding sequence.
  • Substantially pure lichenase can be purified from cell-free medium of such cultures using the methods provided herein.
  • the lichenase expressed by Orpinomyces PC2 has an extracellular (secreted) enzyme having a molecular weight of about 26 kDa, while the recombinant enzyme expressed and secreted by Escherichia coli has an apparent molecular weight of about 27 kDa.
  • the lichenase of the present invention has no apparent activity when assayed with carboxymefhylcellulose as substrate.
  • Figure 1 shows protein staining patterns for crude enzyme sample (lane 1) and purified enzyme (lane 2) and the lichenase activity patterns for the crude enzyme sample (lane 3) and purified enzyme (lane 4) using lichenase as the substrate.
  • Figure 2 is a photograph of crude (lane 2) and purified recombinant lichenase (lane 3) from the extracellular medium of the E. coli culture producing the lichenase. Lane 1 contains the low molecular weight standards.
  • Figure 3 shows the effects of pH on the activity of the Orpinomyces lichenase. Maximal activity on the curve is defined as 100%.
  • Figure 4 illustrates the pH stability of the Orpinomyces lichenase.
  • Figure 5 shows the effect of temperature on Orpinomyces lichenase activity. Maximal activity is defined as 100% .
  • Figure 6 shows thermostability profiles for Orpinomyces lichenase at selected temperatures.
  • Figure 7 is a photograph of a thin layer chromatogram of the products of barley 3-glucan and lichenan incubated with Orpinomyces lichenase.
  • Figure 8 A shows the protein staining profile for low molecular weight standards (lane 1), crude recombinant E. coli cell extract (lane 2), purified recombinant LICA (lane 3), supernatant from
  • Figure 8B is a lichenan zymogram and Figure 8C is a CMC cellulose zymogram. Lanes are as in Figure 8A.
  • lichenase is used synonymously with endo-/ ⁇ -(l-3, l-4)-D-glucanase; the enzyme code assigned to enzymes having this activity is EC 3.2.1.73.
  • the gene and cDNA encoding the lichenase of the present invention is called UcA.
  • the mature UcA gene product (LICA) has an amino acid sequence as given in SEQ ID NO:2, amino acids 1-216 as expressed in Orpinomyces PC2, or a functionally equivalent amino acid sequence, for example, as given in SEQ ID NO: 2, amino acids -8 to 216, or -29 to 216.
  • lichenases with about 70% amino acid sequence identity to any of the foregoing sequences. These functionally equivalent sequences differ in the proteolytic cleavage site for the removal of the signal peptide.
  • the lichenase of the present invention is distinguished from prior art lichenases from rumen bacteria in that there are no repeated oligopeptide sequence motifs in the present lichenase. Without wishing to be bound by theory, it is proposed that the lack of the repeated motifs contributes to the efficient expression and secretion, with functionally correct signal peptide processing in the Escherichia coli recombinant host cells.
  • the lichenase proteins of the present invention is useful for treatment of animal grain- containing feeds to improve nutrient availability and for treatment of grain (e.g. barley or wheat) in the brewing and fermentation industries to increase carbon substrate availability and to maximize production of desired products.
  • the lichenase coding sequences of the present invention are useful to direct the recombinant expression (in Orpinomyces or in other host cells, including, but not limited to,
  • Escherichia coli Bacillus subtilis, Aspergillus nidulans, Aspergillus niger, Saccharomyces cerevisiae, and Pichia pastoris).
  • a cDNA expression library in ZAPII using mRNA isolated from Orpinomyces sp. strain PC-2 cells cultivated with Avicel and oat spelt xylan as carbon source was screened for clones with ⁇ - glucanase activity on lichenan plates. Initially, 15 positive plaques were identified after screening
  • the complete nucleotide sequence of UcA derived from pLIC6 (1.0 kbp) was determined (Table 4, SEQ ID NO: l). The whole sequence was 971 bp with a G-C content of 28%, and it contained an open reading frame (ORF) encoding a polypeptide of 245 amino acids with a calculated r value of 27,929 (See SEQ ID NO: 2). A typical 18-mer poly (A) tail was found at its 3' end.
  • the putative start codon (ATG) for UcA was identified because there were stop codons in all three reading frames preceding the ORF, there was no ATG codon upstream of the identified ORF, and a typical signal peptide occurred at the N-terminus of the ORF.
  • the G+C content of the ORF of UcA was 35.5 % while that of the 5' and 3' non-coding sequences was extremely low (4.3%).
  • the codon usage for UcA was similar to that observed for other
  • Orpinomyces PC-2 cellulase and xylanase genes 21 codons were not utilized, and there was a marked preference for a T in the third position (53 % of all codons contained T in the third position).
  • mRNAs of anaerobic fungi do not contain a typical E. coli Shine-Dalgarno-like sequence for translation initiation. However, presumably the sequence AGA, 10 bp upstream of the ATG start codon, acts as a weak ribosome-binding sequence in E. coli. This sequence was also found in a xylanase gene (xynA) from N. patriciarum (Gilbert et al., 1992).
  • the deduced amino acid sequence of the protein LICA was compared with other protein sequences in the SWISS PROT and GP data banks.
  • a number of /3-glucanases from mesophilic and thermophilic bacteria, including anaerobic rumen bacteria, with some identity to LICA were found. Greater than 50% identity was found with /3-glucanases from certain Bacillus strains, Clostridium thermocellum, and the carboxy-terminal lichenase domain of the xylD gene of the anaerobic rumen bacterium R. flavefaciens.
  • LICA has 30.6% amino acid identity with /3-glucanase from Fibrobacter succinogenes (Table 1). In contrast, limited sequence homology was found upon comparison with barley /3-glucanase.
  • the secreted enzyme encoded by clone pLIC6 was visualized by the zymogram technique involving renaturation of enzyme activity following separation by sodium dodecyl sulfate (SDS)-poly acrylamide gel electrophoresis (Fig. 1). The results show that a strong polypeptide band detected at 27 kDa exhibited only lichenase but not CMCase activity.
  • the native lichenase from the supernatant of Orpinomyces PC-2 culture was also partially purified.
  • the presumptive 21-amino-acid signal sequence deduced from the DNA sequence contains all of the features normally associated with a signal sequence for secretion (Von Heijne, 1988), including a positively charged lysine (-20) terminal n region and a strongly hydrophobic h region from amino acid residues -18 through -6 (10 out of 13 are hydrophobic; amino acid positions are given relative to the first residue of the mature peptide).
  • the c region of the signal peptide conforms to the "(-3, -1) rule", with small, uncharged threonine and alanine residue at positions -3 and -1 relative to the cleavage site, which is typical for a peptide cleaved by signal peptidase I in E. coli.
  • the partially purified native lichenase from supernatant of Orpinomyces PC-2 culture was subjected to N-terminal sequence analysis.
  • the enzyme had a M r of 26,000 and an N-terminal sequence of GTAWNGLHDVMD, (SEQ ID NO:3) which, with the exception of one amino acid, matched the corresponding amino acid sequence deduced from the DNA sequence.
  • SEQ ID NO:3 an N-terminal sequence of GTAWNGLHDVMD
  • proline is conspicuously absent from - 3 to + 1 regions of prokaryotic signal peptides, but it is not usual to have proline at the corresponding region of eukaryotic signal peptide (Von Heijne, 1986).
  • Another reason for the lichenase N-terminal signal sequence being processed differently may come from "the Charge-Block Effect".
  • a region encompassing the first 10-20 resides of the mature protein is also critical for the initiation of membrane translocation in E. coli (Anderson and von Heijne, 1991).
  • This region normally contains few positively charged amino acids; hence, the introduction of only one or two extra positively charged amino acids can dramatically affect secretion (Li et al., 1988). With much higher numbers of charged residues, a similar blocking effect can be observed in eukaryotic secreted proteins (Kohara et al., 1991).
  • the first 20 amino acid residues of the mature recombinant lichenase contain only one positively charged amino acid (histidine); this makes the processed enzyme effectively secreted. If the cleavage site of the lichenase processed in E. coli was the same as in the fungus, the mature recombinant lichenase would be difficult to export from E. coli, simply because the N-terminal region of the mature chain carries too many positively charged amino acids (3 out of 20 animo acid residues).
  • LICA (25.7 kDa) after removal of a signal peptide of 21 animo acid residues.
  • the lichenase activity of the enzyme was measured from pH 4.2 to 8.6 using lichenan as substrate. A typical pH profile was obtained (Fig. 3), with a broad pH optimum from pH 5.8-6.2, with approximately 80% of maximum activity at pH 5.4 and pH 7.0. The enzyme was stable for at least 24 h between pH 3.4 and 9.8 at 4°C (Fig. 4).
  • the lichenase activity was measured in 50 mM sodium citrate at pH 6.0 from 30 to 65°C.
  • the enzymatic activities of the recombinant lichenase were assayed using lichenan, barley- ⁇ - glucan, laminarin, pachyman, CMC, acid swollen cellulose, puslutan and other polysaccharides and glycosides as substrates and analyzed by the dinitrosalicylic acid (DNS) method.
  • DNS dinitrosalicylic acid
  • the enzyme was specific for polysaccharides with mixed l,3-l ,4-/3-D-linkages (lichenan and barley ⁇ -glucan) and did not hydrolyze the other substrates tested (Table 3).
  • K m and V max values at 40°C were obtained from Lineweaver-Burk plots. K, judgment values of the enzyme towards lichenan and barley- ⁇ -glucan were 0.75% (w/v) and 0.91 % (w/v) and V max values were 3,786 and 5,314 U/mg protein, respectively.
  • Lichenase and cellulase activities were detected using zymogram technology with an overlay containing lichenan or CMC, respectively.
  • a clear strong band of lichenase activity was observed at approximately 27 kDa for the cell extract of the recombinant E. coli, the purified recombinant LICA, and the supernatant of Orpinomyces PC-2 culture. No activity was observed at this molecular weight when CMC was used as substrate, indicating that the LICA is specific for lichenan (Fig. 8). Additionally, the results revealed that the UcA gene product was actively synthesized and secreted into medium of the Orpinomyces PC-2 culture.
  • Neocallimastix EB188 appears to lack a lichenase gene.
  • l,3-l,4-/3-D-Glucanases cleave 1 ,4-/3-glycosidic linkages that are adjacent to 1,3-/3- glycosidic linkages in mixed-linked glucans, which comprise an important component of plant hemicellulose.
  • the present l ,3-l,4-/3-D-glucanase (lichenase) does not cleave the /3-l,4 glycosidic bonds in carboxy methylcellulose.
  • 1 ,3-1,4-0-D-glucanase has been found only in certain bacterial strains and in plants.
  • Orpinomyces LICA does not contain a repeated peptide domain, which indicates that it is a free enzyme and not a component of the multienzyme complex. While 1,3-1,4- ⁇ -D-glucanases from the rumen bacteria R. flavefa ⁇ ens (Flint et al., 1993) and F. succinogenes (Teather and Airflow, 1990) have a repeated docking domain, the partial sequence identities between the lichenases of the rumen bacteria and the present lichenase are much lower than those between the present lichenase and lichenases of Bacillus strains or Clostridium thermocellum.
  • the N-terminal signal sequence of LICA is a secretory signal that is functional both in E. coli and in Orpinomyces, the cleavage sites are different. Besides the difference between E. coli and eukaryotes with respect to signal peptides and proteases as discussed hereinabove, the different cleavage sites in LICA signal sequence may also relate to the cell membrane of the anaerobic fungi. Anaerobic fungi lack the ability to synthesize some common cell-membrane constituents such as sterol because of the absence of molecular oxygen. Instead, unusual lipids synthesized by the anaerobic pathway are incorporated into the anaerobic cell membrane (Kemp et al., 1984).
  • NeocalUmastex EB188 does not appear to have a lichenase with the same properties as the lichenase disclosed herein. Since cellulases also have activity to hydrolyze (1 , 4)-/3 bonds in lichenan and /3-glucan, the selective advantage for Orpinomyces to synthesis lichenase in the rumen ecosystem is not clear .
  • the LICA signal peptide of this invention may be used to increase yield of foreign genes in host cells in which they are expressed. Any host cell in which the signal sequence is expressed and processed may be used.
  • the signal peptide sequence (see SEQ ID NOs. 4 and 5 for coding and amino acid sequences) from the Aureobasidium xylanase can be substituted for the exemplified LICA signal sequence.
  • Preferred host cells are Aureobasidium species and S. cerevisiae, as well as other yeasts known to the art for fermentation, including Pichia pastoris (Sreekrishna, K.
  • the coding region for both the signal peptide and the mature LICA protein may be expressed in such hosts.
  • the LICA mature protein coding region isolated from the signal sequence may be expressed in such hosts, or the coding region for the signal peptide isolated from the mature protein coding region may be expressed in such hosts.
  • vectors suitable for transformation of the host are prepared with the gene under control of a promoter expressible in the host, preferably S. cerevisiae.
  • the promoter is a constitutive promoter such as the yeast enolase promoter (Sangadala et al., (1994) "Preparation and characterization of the site-directed E211Q mutant of yeast enolase," In: Abstracts of University System of Georgia 1994 Research Symposium: Advances in Biotechnology, Georgia State University, Atlanta, GA, USA) or a strong inducible promoter such as the yeast alcohol dehydrogenase promoter (Pacitti, et al. (1994), "High level expression and purification of the enzymatically active cytoplasmic region of human CD45 phosphatase from yeast," Biochimica et
  • a constitutive promoter such as the yeast enolase promoter (Sangadala et al., (1994) "Preparation and characterization of the site-directed E211Q mutant of yeast enolase," In: Abstracts of University System of Georgia 1994 Research Symposium: Advances in Biotechnology, Georgia State University, Atlanta, GA, USA) or a strong in
  • the vector is used to transform the host either by integration into the chromosome or otherwise.
  • the host organism is then cultured under conditions allowing expression of the gene and the product recovered from the culture medium.
  • allelic variations may occur in the UcA coding sequence from different strains of Orpinomyces or other fungi which will not significantly change activity of the amino acid sequences of the proteins which these sequences encode. All such equivalent DNA sequences are included within the scope of this invention and the definition of the LICA mature protein coding region and signal sequence coding region.
  • the skilled artisan understands that the amino acid sequence of the exemplified LICA polypeptide and signal peptide can be used to identify and isolate additional, nonexemplified nucleotide sequences which will encode functional equivalents to the polypeptides defined by the amino acid sequences given in SEQ ID NO:2, or an amino acid sequence of greater than 90% identity thereto and having equivalent biological activity.
  • DNA sequences having at least about 70% , 80% and/or 85% homology to the DNA sequences of SEQ ID NO: 1 (nucleotides 210 to 857) and encoding polypeptides with the same function are considered equivalent to the sequences of SEQ ID NO: 1 and are included in the definition of "DNA encoding the LICA mature protein" and the "UcA gene. " Following the teachings herein, the skilled worker will be able to make a large number of operative embodiments having equivalent DNA sequences to those listed herein.
  • codons for conservative amino acid substitutions can change the primary amino acid sequence of a lichenase protein without significantly affecting the function of that protein.
  • conservative amino acid substitutions are well known to the art (See, e.g., Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure, Vol. 5, Supplement 3, Chapter 22, pages 345- 352). Dayhoff et al. 's frequency tables are based on comparisons of amino acid sequences for proteins having the same function from a variety of evolutionarily different sources.
  • a recombinant DNA molecule is not naturally occurring; it is produced by the hand of man in the laboratory. DNA segements or sequences from different sources can be joined by chemical synthesis, enzymatic ligation or by directed recombination.
  • Monoclonal or polyclonal antibodies preferably monoclonal, specifically reacting with a lichenase encoded by a particular coding sequence may be made by methods known in the art. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories;
  • Orpinomyces sp strain PC-2 was isolated and described by Borneman et al. (1989); Neocallimastix sp. EB188 was provided by Dr. Calza (Washington State University).
  • the fungi were grown at 39°C for 7 day in 2 L round bottles, each containing 1 L of basic medium (Barichievich and Calza, 1990) and 0.3 % of coastal Bermudagrass (CBG). The medium was autoclaved for 30 min, and then cooled under a stream of C0 2 .
  • Penicillin (334 U/ml), streptomycin sulfate (80 ⁇ g/ml), and chloramphenicol (10 ⁇ g/ml) were filter sterilized (0.22 ⁇ m) (alt 230) and added to the stated final concentrations just prior to inoculation.
  • Escherichia coli XL-Blue, ⁇ ZAPII, pBluescript were products of Stratagene Cloning Systems (La Jolla, CA).
  • E. coli was harvested by centrifugation at 3,200 g for 10 min (Beckman CS-6R). Cell free culture media were used for extracellular enzyme preparation. The cell pellet was washed twice in half the volume of the culture with 10 mM Tris-HCl pH 8.0 and suspended in the same volume of 25% sucrose 5 mM EDTA. The suspension was shaken for 10 min at room temperature. After centrifugation, the cells were suspended in the same volume of ice-cold water, and the suspension was shaken for 10 min at 4°C. After centrifugation, the supernatant was used as the periplasmic fraction. The cell pellet was sonicated to release intracellular l,3-l,4-/3-D-glucanase activity.
  • SDS-PAGE was carried out in Laemmli's buffer (Laemmli, U.K. (1970), Nature 227:680) with Coomassie brilliant blue R-250 (Sigma Chemical Co., St. Louis, MO).
  • Laemmli's buffer Laemmli, U.K. (1970), Nature 227:680
  • Coomassie brilliant blue R-250 Sigma Chemical Co., St. Louis, MO.
  • samples were pretreated by incubating for 1 h at 40°C in sample buffer and proteins were size- separated using SDS-PAGE at 4°C.
  • gels were washed in 50 mM sodium citrate buffer, pH 6.0 with 1 % (w/v) bovine serum albumin (BSA) (McGrew and Green, 1983).
  • BSA bovine serum albumin
  • Lichenase and CMCase activities were detected using the zymogram method of Beguin (1983) with a overlay containing 0.3% (w/v) lichenan or carboxy methylcellulose and agarose (2% , w/v) in 50 mM sodium citrate buffer, pH 6.0.
  • the bands of enzyme activity were detected by staining the agarose gel with Congo red and destraining with 1M NaCl.
  • /3-Glucanase activity was assayed by mixing a 0.2 ml aliquot of appropriately diluted enzyme with 0.4 ml buffer containing 0.4% (w/v) lichenan or barley /3-glucan (Sigma Chemical Co., St.
  • E. coli XL 1 -blue (pBluescript-/ o4) was inoculated into 500 ml LB-ampicillin (50 ⁇ g/ml) medium and grown to an OD ⁇ of 1.5 to 2.0.
  • /3-Glucanase expression was induced by the addition of 1 mM IPTG, and each culture was aerated for an additional 8 h at 37°C.
  • a cell-free supernatant was obtained by centrifuging the culture at 4°C, 7,000 x g for 10 min. The cell pellet was set aside, and the supernatant was concentrated to a volume of about 50 ml by using an ultrafiltration cell (Amicon Co., Beverly, Mass.) equipped with a PM 10 membrane.
  • the concentrated supernatant was dialyzed against 500 ml of 20 mM potassium phosphate, pH 7.0. Ammonium sulfate was added to a concentration of 0.8 M. The solution was centrifuged at 4°C and 20,000 x g for 10 min to remove precipitated material. The clear solution was loaded on a Phenyl Superose 10/10 column (7.85 ml) equilibrated with 20 mM potassium phosphate, pH 7.0, containing
  • 0-Glucanase was eluted with a 200 ml linear gradient of ammonium sulphate, from 0.8 to 0 M, then further with 100 ml distilled water. Fractions containing /3-glucanase activity were pooled and concentrated, and the buffer was changed to 20 mM piperazine-HCl, pH 5.5. The solution was applied to a Mono Q 5/5 anion exchange column (1 ml) equilibrated with 20 mM piperazine-HCl buffer, pH 5.5. The /3-glucanase fractions did not bind to the column, and the enzyme was eluted by applying 5 column volumes of the buffer.
  • the /3-glucanase-containing fractions were pooled and concentrated, and the buffer was changed to 20 mM sodium acetate, pH 5.0.
  • the enzyme sample applied to a cation exchange Resource S column (1 ml). It did not adsorb to the column, and it was eluted out by further passing through 5 column volumes of the buffer.
  • Final purification was achieved by gel filtration over Superdex 75 10/30 column (composite of cross-linked agarose and dextran gel filtration resin, Pharmacia, Piscataway, NJ) equilibrated with 20 mM sodium phosphate, 100 mM NaCl, pH 6.0. Fractions exhibiting /3-glucanase activity were combined and stored at -20°C.
  • Example 7 N-Terminal Amino Acid Sequencing Amino acid sequencing was done with protein bands isolated and purified after SDS-PAGE.
  • the proteins were transferred onto a poly-vinylidene difluoride (PVDF) membrane in a Mini Trans- Blot cell (Bio-Rad Laboratories, Hercules, CA). The transferred proteins were visualized by Ponceau S staining and then excised with a razor blade. N-terminal amino acid sequencing was performed on an Applied Biosystems model 477A gas-phase sequencer equipped with an automatic on-line phenylthiohydantoin analyzer.
  • PVDF poly-vinylidene difluoride
  • the pH optimum was determined at 40°C using the following buffers: 0.1 M sodium acetate (pH 4.2 to 5.4), sodium phosphate (pH 5.8 to 7.8), and Hepes-NaOH (pH 8.2 and 8.6) with increments of 0.4.
  • Enzyme stability at different pH values was determined by measuring the residual activity after incubating the enzyme for 24 h at 4°C at pH 3.0 to 10.2 (glycine-HCl buffer for pH 3.0 to 3.4; Hepes-NaOH for pH 9.0; piperazine-HCl for pH 9.4 to 10.2). For other pH ranges, buffers were the same as those used for optimum pH determinations).
  • the effect of temperature on /3-glucanase activity was determined by assaying the enzyme at temperatures from 30 to 65°C with increments of 5°C. Thermostability was measured by incubating the enzyme in 50 mM sodium citrate buffer, pH 6.0 for 5 min to 24 h at temperatures from 40 to 60°C with increments of 5°C. The enzyme solution was chilled in an ice bath for 5 min and then analyzed by running the standard assay at 40°C. In all these assays, lichenan was used as substrate.
  • K m and V max For determination of K m and V max , suitably diluted /3-glucanase was incubated with lichenan and barley- 3-glucan at concentrations ranging from 0.02 to 1.0% (w/v) under the assay conditions given. K m and V max values were obtained from Lineweaver-Burk plots.
  • Pustulan 0-1,6 0 0 a The following substrates were not hydrolyzed: Avicel, arabinogalactan, mannan, araban, starch, xylan, pullulan, galactan, and Gum arabic (0.35% wt vo ⁇ 1 ), PNP-0-D-xyloside, PNP-0-D-glucoside, and PNP-0-D- cellobiose (1 mM).
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • AN I-SENSE NO
  • Met Lys Ser lie lie Ser lie Ala Ala Leu Ser Val Leu Gly Leu -29 -25 -20 -15
  • GCT GCT CCT GCT CCC GCT CCT GTT CCT GGT ACT 215 lie Ser Lys Thr Met Ala Ala Pro Ala Pro Ala Pro Val Pro Gly Thr -10 -5 1
  • GAT GGA ACT AAG TGG GAT GAA ATT GAT ATA GAA TTC CTT GGT TAT GAT 551
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • FEATURE :

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Abstract

La présente invention concerne une lichenase fongique, c'est-à-dire une endo-1,3-1,4-β-D-glucanohydrolase, sa séquence codante, des molécules recombinées d'ADN comprenant les séquences codantes de lichenase, des cellules hôtes recombinées et leur procédé de production. La présente lichenase s'obtient à partir de PC-2 d'Orpinomyces.
PCT/US1997/017811 1996-10-04 1997-10-03 Lichenase fongique et sequences codantes WO1998014595A1 (fr)

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EP97909941A EP0931153A1 (fr) 1996-10-04 1997-10-03 Lichenase fongique et sequences codantes
AU47435/97A AU721534B2 (en) 1996-10-04 1997-10-03 Fungal lichenase and coding sequences
CA002267078A CA2267078A1 (fr) 1996-10-04 1997-10-03 Lichenase fongique et sequences codantes
US09/286,690 US6103511A (en) 1996-10-04 1999-04-05 Lichenase and coding sequences

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EP1664322A4 (fr) * 2003-05-22 2007-01-10 Fraunhofer Usa Inc Molecule support recombinee pour l'expression, l'administration et la purification de polypeptides cibles
US8404252B2 (en) 2007-07-11 2013-03-26 Fraunhofer Usa, Inc. Yersinia pestis antigens, vaccine compositions, and related methods
US8778348B2 (en) 2007-04-28 2014-07-15 Ibio Inc. Trypanosoma antigens, vaccine compositions, and related methods
US8951791B2 (en) 2003-02-03 2015-02-10 Ibio, Inc. System for expression of genes in plants
US8962278B2 (en) 2005-08-03 2015-02-24 Ibio Inc. Compositions and methods for production of immunoglobulins
US9115201B2 (en) 2008-09-28 2015-08-25 Ibio Inc. Humanized neuraminidase antibody and methods of use thereof
US9809644B2 (en) 2009-09-29 2017-11-07 Ibio Inc. Influenza hemagglutinin antibodies, compositions and related methods

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US7692063B2 (en) 2002-11-12 2010-04-06 Ibio, Inc. Production of foreign nucleic acids and polypeptides in sprout systems
US7683238B2 (en) 2002-11-12 2010-03-23 iBio, Inc. and Fraunhofer USA, Inc. Production of pharmaceutically active proteins in sprouted seedlings
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US9551001B2 (en) 2003-02-03 2017-01-24 Ibio, Inc. System for expression of genes in plants
US9765349B2 (en) 2003-02-03 2017-09-19 Ibio, Inc. System for expression of genes in plants
EP1664322A4 (fr) * 2003-05-22 2007-01-10 Fraunhofer Usa Inc Molecule support recombinee pour l'expression, l'administration et la purification de polypeptides cibles
US9012199B2 (en) 2003-05-22 2015-04-21 Ibio, Inc. Recombinant carrier molecule for expression, delivery and purification of target polypeptides
US8962278B2 (en) 2005-08-03 2015-02-24 Ibio Inc. Compositions and methods for production of immunoglobulins
US8778348B2 (en) 2007-04-28 2014-07-15 Ibio Inc. Trypanosoma antigens, vaccine compositions, and related methods
US8404252B2 (en) 2007-07-11 2013-03-26 Fraunhofer Usa, Inc. Yersinia pestis antigens, vaccine compositions, and related methods
US8945580B2 (en) 2007-07-11 2015-02-03 Ibio Inc. Yersinia pestis antigens, vaccine compositions, and related methods
US9115201B2 (en) 2008-09-28 2015-08-25 Ibio Inc. Humanized neuraminidase antibody and methods of use thereof
US9809644B2 (en) 2009-09-29 2017-11-07 Ibio Inc. Influenza hemagglutinin antibodies, compositions and related methods

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