BRPI0719038B1 - ISOLATED EXPRESSION SYSTEM COMPRISING A POLYNUCLEOTIDE ENCODING A BETA-MANNASE PROTEIN, ISOLATED HOST CELL COMPRISING SAID POLYNUCLEOTIDE, METHOD OF RECOMBINANTLY PRODUCING A BETA-MANNASE PROTEIN AND METHOD OF DEGRADING MANNANS AND POLYSACCHARIDES IN A PLANT MATERIAL - Google Patents

Abstract

BETA-MANNASE FROM THE COFFEE BERRY BULL, HYPOTHENEMUS HAMPEI, AND USES THEREOF. The present invention relates to an isolated Beta-mannanase protein having an amino acid sequence that is 90% similar to the amino acid sequence of SEQ ID NO: 1, as well as isolated polynucleotides encoding the Beta-mannanase protein and isolated expression systems and host cells containing the polynucleotides. The present invention also relates to a method of recombinantly producing the Beta-mannanase protein. Also disclosed is a method of degrading mannans and polysaccharides in plant material by involving the supplied plant material and contacting the plant material with the Beta-mannanase protein of the present invention under conditions effective for degrading mannans and polysaccharides in the plant material.

Description

[001]This application claims the benefit of priority of United States Provisional Patent Application Serial No. 60/866,705, filed November 21, 2006, which is incorporated herein by reference in its entirety.

FIELD OF INVENTION.

[002]The present invention relates to an isolated β-mannase protein and its uses.

BACKGROUND OF THE INVENTION.

[003] Mannanases are enzymes that hydrolyze mannans and related hemicellulosic polysaccharides such as galactomannan and glucogalactomannan (also called galactoglucomannan). These polysaccharides are characteristic components of plant cell walls and thus an important potential commercial use of mannanases is the degradation of hemicellulosic materials from plant biomass, thereby providing a means of recovering soluble sugars from these biopolymers. Mannan polysaccharides are also found as storage polymers in the seeds of some plant species, such as those of leguminous plants and coniferous trees.

[004] In the coffee bean, galactomannans accumulate in extremely high concentrations and represent approximately 24% of the dry weight of the bean (Bradbury et al., "Chemical Structures of Green Coffee Bean Polysaccharide," J. Agric. Food Chem. 38:389-392 (1990)). These polysaccharides consist of a linear chain of mannosyl residues that are linked to each other via beta 1,4 glycosyl bonds, to which are attached the monomers of the alpha-galactosyl residue. It is known that endo-beta-mannanases (EC 3.2.1.78) hydrolyze mannan polymers during seed germination, thus facilitating the exit of the radicle during germination and releasing small oligosaccharides that are then used as an energy source for the growth of the young plant. Indeed, in several plants, it has been shown that endo-β-mannanase activity is primarily detected in the endosperm of germinating seeds (Bewley, "Breaking Down the Walls— A Role for Endo-β-Mannanase In Release from Seed Dormancy?" Trends Plant ScL 2:464-469 (1997)).

[005]Mannanases are produced by microorganisms such as fungi, yeasts and molds, as well as Bacillus subtilis, Aeromonas, Enterococcus, Pseudomonas and Streptomyces. Some higher plants or animals can also produce mannanases; however, there is no report in the literature describing a β-mannanase from insects.

[006]Microorganisms that are typically used for the commercial production of mannanases include Trichoderma or Aspergillus spp.

[007] In industrial processes, during coffee treatment, mannans and their derivatives constitute a considerable portion of the insoluble sediment. Furthermore, during the first extraction stage in coffee production only approximately 50% of the mannans are soluble and these polymers are therefore responsible for most of the secondary precipitations that occur during subsequent stages.

[008]European Patent No. 0676145 A demonstrated that it is possible to hydrolyze coffee galactomannans using an immobilized mannanase extracted from Aspergillus niger.

[009]The present invention is aimed at overcoming these and other limitations of the technique.

SUMMARY OF THE INVENTION.

[010] One aspect of the present invention is directed to isolated β-mannanase protein having an amino acid sequence that is 90% similar to the amino acid sequence of SEQ ID NO: 1. The present invention also relates to an isolated polynucleotide encoding β-mannanase protein and an isolated expression system and a host cell containing the polynucleotide.

[011] Another aspect of the present invention is directed to a method of recombinantly producing β-mannanase protein. This method involves providing a host cell containing the polynucleotide of the present invention and culturing the host cell under conditions effective for the host cell to express β-mannanase protein. The β-mannanase protein is recovered.

[012]A further aspect of the present invention is directed to a method of degrading mannans and polysaccharides in plant material. This method involves providing plant material and contacting the plant material with the β-mannanase protein of the present invention under conditions effective for the degradation of mannans and polysaccharides in the plant material.

[013]The present invention relates to an isolated polynucleotide sequence encoding a mannanase enzyme involved in the hydrolysis of mannan polysaccharides, including unbranched or branched mannan molecules linked to each other via the beta 1,4 glycosyl bond. The polynucleotide is isolated from an insect (coffee berry borer, Hypothenemus hampei) genome.

BRIEF DESCRIPTION OF THE DRAWINGS.

[014]Figure 1 is a photograph showing SDS-PAGE analysis of recombinant β-mannanase protein according to one embodiment of the present invention expressed in recombinant baculovirus. Lane 1: molecular mass markers (kDa); Lane 2: Speed-vac concentrated protein; Lane 3: 6x-His tagged mannanase purified using paramagnetic particles.

[015]Figure 2 is a graph showing the time course of hydrolysis of AZC-galactomannan with soluble recombinant β-mannanase from H. hampei. Enzyme activity was determined by the blue increase in absorbance over time; β-mannanase activity is the mean ± SE for the three reactions.

[016]Figure 3 is a graph showing the effect of pH on the activity of recombinant H. hampei β-mannanase according to the present invention. The buffer was 200 mM sodium acetate with pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0.

[017]Figure 4 is a graph showing the effect of temperature on the activity of recombinant β-mannanase from H. hampei. Enzyme solutions in 200 mM sodium acetate at pH 5.0 are incubated at various temperatures for 120 minutes and activities are measured as described in the Examples (below).

DETAILED DESCRIPTION OF THE INVENTION.

[018] One aspect of the present invention relates to a β-mannase protein isolated from the coffee berry borer (Hypothenemus hampei) having an amino acid sequence of SEQ ID NO: 1, as follows:

[019]The present invention is also directed to isolated β-mannanase proteins having an amino acid sequence that is at least 90% similar, at least 91% similar, at least 92% similar, at least 93% similar, at least 94% similar, at least 95% similar, at least 96% similar, at least 97% similar, at least 98% similar, and/or at least 99% similar to the amino acid sequence of SEQ ID NO: 1.

[020] The polynucleotide encoding the β-mannanase protein of SEQ ID NO: 1 has a sequence of SEQ ID NO: 2, as follows: tgctgtaccc ggattcacgg 120tttccggtac tcgaattcca gatgctaacg gtcaggaatt tatgataage ggggtcagtc 180acgcacatac etggtataag gatgatatta atggggccat cacatccatc getgetgetg 240gcgccaacac ggttcgcatt gtactttcta atggcggaca gtggacaaaa gacaacetgg 300attcagttca gaacattetg tecctctgtg agagccataa gettattgee atgetggaag 360ttcacgatgc caccggαaat gacagcαaag aaacaetgga aaatgccgtg aattactgga 420aagagetteg ggacttgctc attggtaagg aagacagagt tattatcaat atagecaatg 480agtggttcgg tacctgggat actgctggct gggctgaegg ttátaaagtt gtcattccgg 540aagtacgtaa cgccggactg gaacacctgc tggttgtaga cacagcggga tacggacaat 600atcctcaagc tatttttgaa aaaggtaagg aggttttcca gacagacctt cttgcccgca 660cggtgttttc cattcacatg tatgaatatg cagcgacgga tgtaacaatg ataaaaggaa 720atattgactc ggccttgaat acaggcatcc cggtgattat tggagaattt ggtgaccgaa 780aaccggagtc gcagcatgtt gatatcgata ccatcatgag ctacactcgg gagaaatccg 340taggctggtt ggcctggtcc tggtacggta acgqtaacga tgaatcaatt cttgacctga 900cgaacggacc tagcggaqat tacagtctta ctaacgtggg gagtcaaatt gttgacagtg 960agaacggcat tcgcaaacc tccacaatct gttcaatatt caattaaaaa aaaagatgtt 1020tgtttgtgca tttttgttat aataaacgtt tcatttgcat att 1063

[021] Isolated polynucleotides that have at least 90% similarity, at least 91% similarity, at least 92% similarity, at least 93% similarity, at least 94% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity, and/or at least 99% similarity to SEQ ID NO: 2 are also encompassed by the present invention.

[022] The genomic sequence from which the isolated polynucleotide of SEQ ID NO: 2 is derived has a sequence of SEQ ID NO: 3 as follows: atggttgagt tcaccaatca agaatatgca gacatgcatt tgatttatgg ccaagccaat 60ggcaattcct acgaagcgcg cagactctac gcacgtagat atcctaatcg gagactacct 120gatccaaaaa catttccaaa tattcacatt cgactatgtg aaactggaac atataaacag ISOttcagtggtt tcgaaggagt acatcaaatc gcgagaactc cagaaatcga agaagccgtt 240ctaaatagtg ttgaagccga tcctgctacg agcacaagga aaáttgcaat aacattgaac 300atttcattta tgcttgtctg aagaattctg actgataacc ttttgtatcc ttaccacctt 360acaagggttc aagctcttct cccacgagac tttcctttag gcgtaaattt ttgcgagtag 420ttctttacaaa tgctggctca aaatccgtcg tttgcatcgt ttgcgtcgtt ttgtttattt 400 tatttacgga tgaagcaaat ttttcaagaa attccatccg aaattttcat aatgaacatt 540tttggggaga agaaaatcca catttagtac gagaaaacaa ttttcaacat caattttctg 600tcaacgtttg ggcaggaatt attggcgatt atttaatagg accatttttt ctgtcgaaga 660ggttgaatgg tggctattat catcggtttt tcgaagagga acttcccgta cttttagatg '.'20aggtaceget tcttttgaga aaccaaatgt ggctaatgca cgatggtgcg ccagtccatt 780ttagteggga agtaagggag ttcctaaatg aacattatca caaccgttgg attgatcgag 840ggggaactca gtcatggccc ccgaggtccc cgcacctgaa tagtctggat ttttttttct 900agggacatct caaatcettg gtgtaccaaa ccgcaattaa cacagtggag gaattgcgaa 960acagaatagt egattcatgt aacgtcattc gcaatactce tggtattttt gaaagagtcc 1020gccggtctat gaggcacaga gcggaatctt gcatcttagc aagaggagga cattttcaac 1080agttcctata gtcttqtttt atttagatta aattactttt actgttacct tacgaattta 1140atacataaga ttcattgtac tcttttgttg tacgrtttcc ttaatatgca tcggtaactg 1200tttatgcaaa tttttcgcaa atgataaaag atacgagaaa aatgcaagag atcaaaaagt 1260aagagaaata gacaaggaat ctaaatgtga aatcaaaatt tatacatagt gttccaaaaa 1320aaagttagga agcaaaaaat agcacatgac ccaagaaaac ataagaccct gtatatggag 1380agçaacgatt ttgccatttt catatagagg ctcttaaaaa atatagatat accaaattte 1440attaaattat ctttaggcat aagaaagaaa atagtagaaa atttaaaaaa aatcaaactt 1500tatcaccctg tatatcaaaa atggtgcgtt tttccccata ggtgtattag catttttttc 1560ttattttgcc gaatactatc accccctgaa atatctccat gatgatctgt tacaccctgt 1620acacctaaaa agtaaaataa taaaacgttt aaattttatc ttttaacgta gataagattt 1680tgcgtccttt gtttccttct aagttttaat cgagatttcg cctcattttc gctcattcgc 1740cagaagacct cagtgaaagc gatbcattaa gtctgaaatt taactttgtt ccctaccgaa 1800tattcttttt ctgacgatag acgatagctg atcgggtgtg tactcaattc tttaaggagt 1860ttacaatatg accgctgata cattaacgcg ggcactgctg ctgttgctgt tgttgcgcgc 1920 tgctgctgct gtacccggat tcacggttt-c cggtactcga attttagatg ctaacggtca 1980ggaatttatg ataagagggg teagtcacgc acatacctgg tataaggatg a tat taatgg 2040ggccatcaca tccatcgctg ctgctggcgc caacaeggtt cgcattgtac t ttctaatgg 2100cggacagtgg acaaaagaca acctggattc agttcagaac attctgtccç tctgtgagag 2160ccataagctt attgccatgc tggaagttca cgatgccacc ggcaatgaca gccaagaaac 2220 actggaaaat gccgtgaatt actggaaaga gcttcgggac ttgctcattg gtaaggaaga 2280 cagagttatt atcaatatag ccaatgagtg gttcggtacc tgggatactg ctggctgggc 2340 cgacggttat aaagttgtca ttccggaact acgtaacgcc ggactggaac acctgctggt 2400tgtagacaca gcgggatacg gacaatatcc tcaagctatt tttgaaaaag gtaaggaggt 2460 tttccagaca gaccttcttg cccgcacggt gttttccatt cacatgtatg aatatgcagα 2520 gacggatgta acaatgataa aaggaaatat tgactcggcc ttgaatacag gcatcccggt 2580 gattattgga gaatttggtg accgaaaacc ggagtcgcag catgttgata tegataccat 2640 eatgagctac actcgcgaga aatccgtagg ctggttggcc tggtcctggt acggtaacgg 27Q0 taacgatgaa tcaattcttg acctgacgaa cggacctagc ggagattaca gtcttactaa 2760cgtggggagt caaattgttg acagtgagaa cggcattcgc aaaacctcca caatctgttc 2820 aatattcaat taaaaaaaaa gatgtttgtt tgtgcatttt tgttataata aacgtttcat 2880 ttgcatarta aatatactaa tccaatatat atttatagac aatagattat taaaaaagta 2940 aattttaaaa taacttcttc aaaaaagaac atttacgctc aaagtgacct atagacgtca 3000 ataatttaaa atgtcactct tcgcacattg acaataacct gcatagacgt ctatgaacgt 3060 cgttgtctat agacgtgttc ctttaattgt tttctaaagc tttgatcaat tggttcagaa 3120aaacggttca atagattcat ttaataattt acaggactat tgggggtaca ttaggctata 3180 aaacggcetc tcaatatttg tcttc^catc aatatttaaa aagtaatagt agatttgtta 3240 aaggactgta aaatgtaatt ttttagtagt ttttccaaat taaagctaag agtaaaaaaa 3300 acggtttttc tacaaaagtc atggaagggt tttgtaggga atttaatcag gtttttaaaa 3360 ctatccttga aattaaagtt tacttaagcg atcactggtt gctgagatat cgatgatcaa 3420 agataaaagg atcctttttc tttcaaagtt agatgtctca gcaagggatt gacgtagatg 3480 tatgaaaaaa aaaacaaaat gaagctgaat aaacaaggta aecgaccact gtacgacaag 3540 ggttcaaatg gaaaaaaatt tdgagacccz atggagactt tagaagaaga agaaaatLLt 3500 gaaaaatgct tacctcgcgc cattccttgg gattgcgcgg taaccataac tccaaaggaa 3660 attcegatgt aagatctgaa aactataaaa cattaagctt caaaatgctt ttttctccaa 3720 ctcgatacga cúgttttttc acaaagatac ∑caaagaaca c∑aaaaaaaa taaaaaaagg 3760 tttttacttt aattttttgg attattatta ∑taacattat ttaatctaaa ataatactga 3840 tattggtatt aactttcacc aggtacactg gtttcaatag aaacgttacc aatttagtta 3900 catagcatca aagaaaagaa tgacattatg atcatcaatt ataattgat” gttcgattat 3960aatataacta ttattgatta ttatattatt ataactctct taggtattaa gcccttaagt 4020caaaaatcgt agttttctac aaaacgagat taaaaatttt ataacgctat gcaacagaaa 4080aaaaaattca ctgggtttac agtacgtggt gatgatacct cactatttac tcaattaata 4140tttatatata aaatagtccc atqaattatt taaattttca aaaaaaaaat ttattaaatt 4200gttcaacsaa gagttaacaa taatttcaca atagttaaga actaatttct taattttcaa 4260tatggccccc ttcctgtaaa achacttat ctattcgttt tctgatgttt tgcacaactt 4320 aaataataaa tgttgtaata tttcttgaaa atttaattaa ggttaccaga tgtcactttt 4380 ttacaattat tctaacaaga gttgagtgat agtttaggg∑ tcgatccctg ctacctccga 4440 tatttttttt tttcgttttt ttuttgttaa taacaatagt aataattgtc aaaataatta 4500 aaaacgataa aaataattta tttgaegcat tttacagtta tttaaagctt gtaatgagag 4560aatttacatg attcgcatat taaattaagg attctcacta caaatttcat atttcaaaaa 4620 caattggt.ec tattttaata aaattateta ccaggaggtt tttgatgatg ctetttcata 4680 atatgttaaa aaaatgcgtt taaaattacc ctaatacatt tttctgtaaa atctaccota 4740 atatttaact ataaaaaegt acgccaatga cgaagggaaa ccattttagg caaatcacaa 4800 teggaattea aagatacaca actgatccaa atttgaagtg aateggetaa acagtttttg 4860 agatacaasa gtggctccat gaategtgeg acatactata tgcgatcaaa ataagaettt 4920 tttttcctat aaacatgtgc cctaaaatgc accccctcca aactacagcç attctaagtt 4980 gegegacaaa aatcaattat tttaaatttt gactacagtt atggatcaaa tttttgaaaa 5040 attgcacatc cgtttttctt aaacactgta ttcatttctt gctttaaaaa taacgtaacc 5100 ctaattttag agaagatgga gagggatcca cttattgegt aaataaaatt taatgtttcc 5160 aacgagaaat cctaacttta accatoagca tctagtagac catggaaaaa tctaaaattt 5220 atctctagca ttttcttaat gcaactaatc gaatctttac aggacctgac ataaaaaaat 5280taattgatga tacctctttt ttatcaagtc tcatttacat agaataacag gcatgattag 5340 catttgttga cgtcacaaaa aattttctcg gcaattacaa atqaacagat ttctgtgaaa 5400 aaattaattt aatgttgaat gcctatcaga aattagggtg caatatatca ctgaaaattc 5460 atttcttgca atatcactta agttttttcc gβaaaatatg gattcagtta gtaatgaact 5520 aggtatatgt gcggtaaatt tatgttgcaa aaaaatgtac gcagattacc tgaattataa 5530 caacaaagtt gtcgaaaaat gtcgaaacac ctccgaaaat cgaaacatag caaaaattat 5640ccgagtcatt tatgcacttg aattcgtgat ttcttttact catttcacaa atttattaca 5700 tgattaaaat taaattattt attaaattac aagagaatga taaaaaaaat aattaaggct 5’60 tttaaatgtt gtatatgaac tgtcacaccg taacagattt gtcaaeatat taacaattga 5020 cagtaaaaat ttcaaattta taattcggtc atccattaga aaattcataa cttcactatt 5880 tatccataaa tttgcatcca aagtaacttg ttcttttcat gaatgtgtca gctctaçata 5940 aaacaattga aaetggacct atctttgcga tttctatttt ttccagggcc atatgggaaa 6000ctttgcaata aatcttgtgc aataaattat gacaattagg aatatttggt gcggtgtaca 6060 tattatatac aggttgaaga aaatacctcc cccataaaag qgccttcaaa atagtctaat 6120 acaattttgc ccttaaacga acagggaaga taatattaaa ggatattgaa atttatatgg 6180 gttttcctat tgtcggtccg gaggggcaac atccaatata ctatatgaaa taaagttgtc 6240 tatagttagt acattgttaa taatttgatt ttcatatatt ctgtattcag tttctaccgt 6300 acaagtagcg gcggagatgt ggatgtcatg tattattaaa cttgtatgga aaaaaaggat 6360 aaaaaaggac acttatatct ccaccattga tgaatggtta aagctggtaa ttttttggaa €420 tacaectacc gatatgctaa actcatatac gaaatttggt taaaaaatct taagccgttt 64$O cggaatttaa aaaataaaga aatgttcttt tttttaaact ttaacaccct gtatctcgga 6540 aacggtgcgt tigcgggccc atgttcatat aaactttttt gcttattttt gtctgaagaa 6660 tcatcccttg aaatttatgc acgtacttaa ttaacaccct gtatttcacg taccatgtcc 6660 tactatacct aaactcaaca gttggcgaga aattttgttg tcgtacctgg tttttgggcc 6720atcctgtata catggattta tcatcacaaa aacctcaacc aaaaataaaa artgacgtgt 6780 atgagcgttt tttttttttg aaaaacaaat ttctgaattt taaatgaatg tattccttat 6040 tttatcataa acctgtatat aaatttaaaa aaaacgtgat gtgatacgtg aaaactaagt 6900 tgctatttgg attcagαagg acaaaaLtta tatgttttat ctaaaattat gcaaaaaata 6960 ttttcatcgc agacccgtgt tatcaatgta aaaattaatt aaacccoctc gtaaaagccs 7020 gcgttttgac atttgaatgt ttccgcccca atgttgaagg gasaaataaa aagttactag 7030aatgtaacta gttaggctgc catatttgga gtaacatgtt ccctctctct ctctaacaca 7140 cgtgaacata actttgcggt actgtataga tttagtgact gtacctacat agtcatacgt 7200 atggaaactt wholesale aatccgtttt cacttatccc ctcgccccca accaccccaa cgttcagaat 7380 ttcaaatggc accatctgtc atgtaatacc tcaaatggaa ggtatttcaa aactgcattt 7440aggggtataa ttagatttta atttattgat tcgtttttga gaaaaagtgc ctcaaaaagg 7500 taaaattaaa aattttaata ttgtttctta caaaaacaat taggttttcaatattagttg 7560 ttaggaacag ggccggattt agggcagggc aggcggggct actgcccegg ggcctccaca 7620 aggaaggggc ccccacaata gagaaaaata aatatcgaae tttcgacggg tcagcaactt 7630 ttaatttttt ttagtttttt caatacttaa gtgtctttcg ggacccgtgg agcgaattgg 7740 ggtgctgggt ggggtaaaca actagctccg agtgtgctaa ttgcggcgtt ctttaaaaβa 7600 aataatttat ttttgtttct ttttttcatt ttctggcgtt tcetggatgc caaaaatatg 7860 ttttttttct caaaactgta aatgtcgtta aagcgttacg gaccccaagt ttccatttac 7920 tgtcaatttt ttattttatc aagcatctcg gtggcaatac ttttttttta atstgtaatt 7900 tgtaatttgt gctcatgagt taaaagaaa taaaataagt aaaaagaaat tccaaggttt 8040 tataaaaagt atatatggta accgggtgac tattaaaatt tcttctcaag taggcrttgg 8100 aattatttga caccgagtcc gtatgtctta gatttattaa cagattcgta tttaagtgag 3160gttgggttta tttgtttata ttcgaaacta agtaaagaac atatcaaaga cttacggact 0220 cgctgtcaaa taattccaca gcctacttga gaagaaattt taatagtcac ccgttataaa 8280 tacgatttaa atettacggt tggcaacact gtcgtggtta agaagtgtcc gagtgtagcg 8340 tgctcgaagc ggggaatccc taaattttat tagaagctac ggacgtattg caacaaaagt 8400 aaaatcttct gaatttatat ctacaaacac acgtaagtat tttcgtattt atcttgtgat 8460 ctataagata taatgaattt tar.attt.cat tgtgcaatta cgaaaatttg tttttttttc 8520ttaaaggagc gatatgcttt aasgagcaaa aacgteaaca gaggttatgg aggttttatt B580 ccacgactac attcttattt tcttaagatt tcttttacaa tggtattatg agtgatcgcc 8640 ataaaactcc tcgagttttc gctagtggat cccaaaaaag gaaattgcaa acagaacgtg 3700 aaaaaaaaa agtgaagaaa atttagctaa aatacccaaa ttgaccaact attttacatç 8760 gacacccaaa caaaaacttc cgcaagatcc tgaaaaatca gcagaagatt csgcagtaga 8820 tggagatggg gttgatagta atcaagataβ tccagctgtt acatcaggcg acaccatagg 6800 atcttcaaaa acttgtagtc acaatcaaga ggaagtagat tttcgtggtt tcaaaaatga 8940cattggtett tggcctgacg tcataacaga agaaatgatc aaatattggg cgaagaaggg 9000 ttccacaaaa ctgcaaaact gtgatgaagt ttctctgcag aattcagttc tccaagacca 9060 gtcgcaagat aataaaaact ttgttcggaa atgttcaaag aatatgttta cacgtcgcaa 9120 tcaaaatcaa gagactgtta atcgattctg gctttgtttt tctccaacta agggaaaagt 9180 atattgctat gcatgtaaat taatgtccac tcaaaaacga agctaagtgg ggaaggcttc 9240 agtgactgga aacatgcatc tgagcggctg tacgagcatg agatttcaaa aactcatttg 9300 gaatcagtga tgaatttagt gcaaegagga gaagtcacag gacgtatcga tcaagagtta 9360 acgatacaag aggcacaaca aattgaatat tggcgaaaaa ttcttacaat tgtcgtcagt 9420 acgattaaat tcattgetga acgcggatta gcctttcgag gagacgatga aattattgga 5480 tcatcgagaa atggcaattt tctggαtatt ttagaattgc tagccgagta cgaaccaatc 9540 ttggcagctc atttaaaaca gcatgcaaac aaagggagag gtcacgtcaa ttatctttct 9600 tctacgatct gcgaagaact gacaaattcc atgggtgatc aagtgttcaa tgaaatcgta 9660 gcaaggatta aaaaatcaaa gtactattct gtttcagtgg actctactcc tgacgaatct 9720 catatcgatc aacttactat agttattcgc tatattgaag gatcgatgcc aaaggaacga 9780 tttcttattt tttaccaaat tgcggtcata ctggtgaagc cacagcaaaa gctttactac 9840 aatttttaag ttaecatcaa attgacatec ttaattgccg aggtcaatcg taegacaatg 9900 ctgcaaatat gagtggtaaa tatcaaggga tgcaagctct rattttgcag aaaaatcatt 9960 tatctacgtt. tgtaccatgt tgtggtcact cactcaactt agttggaaag gcagctgcta 10020 actcttgtgc atcggcagtt caattctttg atttcgttca gaatttatat acgtttttta 10080 cagcaagtac acaacgatac cgaattctgt ctgaaaaatt atcagagaaa aaaagcggac 10140 agtcatatgt tttaaaaaat cttagcgata ctcgctggtc atgtagggtt gcagccacga 10200 aggccattgt tatgggatat tctgaaatcg aagaagctct aaccagcata tcttctgata 10260 aggaacagaa agat 10274

[023] The determination of percent identity, that is, sequence similarity, between two amino acid sequences or two nucleotide sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin et al., "Methods for Assessing the Statistical Significance of Molecular Sequence Features by Using General Scoring Schemes," Proc. Natl. Acad. Sci. 87:2264-2268 (1990), which is incorporated herein by reference in its entirety, as modified as in Karlin et al., "Applications and Statistics for Multiple High-Scoring Segments in Molecular Sequences," Proc. Natl. Acad. Sci. 90:5873-5877 (1993), which is incorporated herein by reference in its entirety. Another non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm of Myers et al., CABIOS (1989). Such an algorithm can be incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis et al. "ADVANCE and ADAM: Two Algorithms for the Analysis of Global Similarity between Homologous Informational Sequences,” Comput. Appl. Biosci. 10:3-5 (1994), which is incorporated herein by reference in its entirety, and FASTA described in Pearson et al., "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad. Sci. 85:2444-8 (1988), which is incorporated herein by reference in its entirety.

[024] The isolated β-mannase protein of the present invention is preferably produced in purified form by conventional techniques. For example, to isolate the protein, a protocol involving a host cell such as Escherichia coli can be used, in which the E. coli host cell carrying a recombinant plasmid is propagated, homogenized and the homogenate is centrifuged to remove bacterial residue. The supernatant is then subjected to sequential precipitation with ammonium sulfate. The fraction containing the β-mannanase protein can be subjected to gel filtration on an appropriately graded dextran or polyacrylamide column to separate the protein or polypeptide. If necessary, the protein fraction can also be purified by HPLC. The isolated β-mannase protein of the present invention can also be produced according to a protocol involving insect host cells, preferably the insect Sf9 cell line.

[025]The present invention is also directed to fragments of the β-mannanase protein of the present invention. Fragments of the β-mannanase protein can be produced by digestion of an entire protein with proteolytic enzymes such as chymotrypsin or staphylococcal proteinase A, or trypsin. Different proteolytic enzymes will likely cleave the β-mannanase protein at different sites based on the amino acid sequence of the protein.

[026] In another approach, based on knowledge of the primary structure of the protein, fragments of the genes encoding the protein can be synthesized using a PCR technique in conjunction with specific primer kits chosen to represent certain portions of the protein of interest. These would then be cloned into an appropriate vector for the expression of a truncated peptide or protein.

[027] Chemical synthesis can also be used to make suitable fragments. Such synthesis is carried out using known amino acid sequences for the protein being produced. Alternatively, subjecting a complete β-mannanase protein of the present invention to high temperatures and pressures will produce fragments. These fragments can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE).

[028]Variants may also (or alternatively) be made, for example, by the deletion or addition of amino acids that have minimal influence on the properties, secondary structure, and hydropathic nature of the protein. For example, a protein may be conjugated to a signal sequence (or leader sequence) at the N-terminal end of the protein that co-translationally or post-translationally directs the transfer of the protein. The protein may also be conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the protein.

[029] The protein of the present invention is preferably produced in purified form (preferably at least about 80%, more preferably 90%, pure) by conventional techniques. Typically, the protein of the present invention is secreted into the growth medium of Helicobacter cells or host cells expressing a functional type III secretion system capable of secreting the protein of the present invention. Alternatively, the protein of the present invention is produced but not secreted into the growth medium of recombinant host cells (e.g., Escherichia coli). In such cases, to isolate the protein, the host cell (e.g., E. coli) carrying a recombinant plasmid can be propagated, lysed by sonication, heat, differential pressure, or chemical treatment, and the homogenate is centrifuged to remove bacterial residue. The supernatant is then subjected to sequential precipitation with ammonium sulfate. The fraction containing the polypeptide or protein of the present invention is subjected to gel filtration with an appropriately graded dextran or polyacrylamide column to separate the protein. If necessary, the protein fraction may also be purified by HPLC.

[030]The present invention also relates to an isolated polynucleotide encoding the β-mannanase protein and an isolated expression system and host cell containing the polynucleotide.

[031] Another aspect of the present invention is directed to a method of recombinantly producing β-mannanase protein. This method involves providing a host cell containing the polynucleotide of the present invention and culturing the host cell under conditions effective for the host cell to express the β-mannanase protein. The β-mannanase protein is then recovered.

[032] The polynucleotide of the present invention may be inserted into any of many available expression vectors using agents that are well known in the art. In preparing a DNA vector for expression, the DNA sequence may typically be inserted or substituted into a bacterial plasmid. Any convenient plasmid may be employed, which will be characterized by having a bacterial replication system, a marker that allows selection in a bacterium, and usually one or more unique, conveniently located restriction sites. Several plasmids, referred to as transformation vectors, are available for plant transformation. Selection of a vector will depend on the preferred transformation technique and the species targeted for transformation. Several vectors are available for stable transformation using Agrobacterium tumefaciens, a soil-borne bacterium that causes crown blight. Crown blight is characterized by tumors or rashes that develop on the lower trunk and main roots of the infected plant. These tumors are due to the transfer and incorporation of a portion of the bacterial plasmid DNA into the plant chromosomal DNA. This transfer DNA (T-DNA) is expressed along with the normal genes of the plant cell. The plasmid DNA, pTi, or Ti-DNA, for “plasmid-induced tumor,” contains the vir genes necessary for the movement of the T-DNA within the plant. The T-DNA carries genes that encode proteins involved in regulating the biosynthesis of plant and bacterial nutrients (opines). The T-DNA is bordered by two imperfect 25-bp direct repeat sequences called “border sequences.” By removing the oncogene and opine genes and replacing them with a gene of interest, it is possible to transfer the foreign DNA into the plant without tumor formation or multiplication of Agrobacterium tumefaciens (Fraley et al., "Expression of Bacterial Genes in Plant Cells," Proc. Nat'l Acad. Sci. 80:4803-4807 (1983), which is incorporated herein by reference in its entirety).

[033]Further improvement of this technique led to the development of the binary vector system (Bevan, "Binary Agrobacterium Vectors for Plant Transformation," Nucleic Acids Res. 12:8711-8721 (1984). In this system, all T-DNA sequences (including boundaries) are excised from pTi, and a second vector containing T-DNA is introduced into Agrobacterium tumefaciens. This second vector has the advantage of being replicable in E. coli as well as A. tumefaciens and contains a multiclonal site that facilitates the cloning of a transgene. An example of a commonly used vector is pBin19 (Frisch et al., "Complete Sequence of the Binary Vector Bin 19," Plant Molec. Biol. 27:405-409 (1995), which is incorporated herein by reference in its entirety). Any currently known or subsequently described suitable vectors for genetic transformation is suitable for use with the present invention.

[034] Suitable vectors for practicing the present invention may also include, but are not limited to, the following viral vectors, such as the lambda gt11 vector system, gtWES.tB, Charon 4, and plasmid vectors, such as pBR322, pBR325, pACYC177, pACYC 184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClO1, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) which is incorporated herein by reference in its entirety), pQE, pIH821, pGEX, pET series (Studier et al, "Use of TJ RNA Polymerase to Direct Expression of Cloned Genes", Methods in Enzymology 185:60-89 (1990), which is hereby incorporated by reference in its entirety) and any derivatives thereof.

[035] U.S. Pat. No. 4,237,224 issued to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced via transformation and replicated in single-cell cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture.

[036] Various host vector systems can be used to express the protein-coding sequence(s) of the present invention. Primarily, the vector system must be compatible with the host cell used. Host vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with viruses (e.g., vaccinia viruses, adenoviruses, etc.); insect cell systems infected with viruses (e.g., baculoviruses); and plant cells infected with bacteria. The expression elements of these vectors vary in their potency and specificity. Depending on the host vector system used, any number of suitable transcription and translation elements can be used.

[037] The protein according to the present invention may be incorporated into an appropriate vector in the sense direction such that the open reading frame is properly oriented for expression of the encoded protein under control of a promoter of choice. This involves the inclusion of appropriate regulatory elements in the DNA vector construct. These include untranslated regions of the vector, the useful promoters of the 5' and 3' untranslated regions that interact with the host cellular protein to effect transcription and translation. Such elements may vary in their potency and specificity. Depending on the vector and host system used, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.

[038] A constitutive promoter is a promoter that directs the expression of a gene throughout the development and life of an organism. Examples of some constitutive promoters that are widely used for inducible expression of transgenes include the Agrobacterium tumefaciens nopaline synthetase (NOS) gene promoter (U.S. Patent No. 5,034,322 issued to Rogers et al., which is hereby incorporated by reference in its entirety), the cauliflower mosaic virus (CaMV) 35S and 19S promoters (U.S. Patent No. 5,352,605 issued to Fraley et al., which is hereby incorporated by reference in its entirety), derivatives of any of several actin genes known to be expressed in most cell types (U.S. Patent No. 6,002,068 issued to Privalle et al., which is hereby incorporated by reference in its entirety), and the ubiquitin promoter, which is a known gene product by accumulating in many cell types.

[039] An inducible promoter is a promoter that is capable of directly or indirectly activating one or more transcriptional DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequences or genes will not be transcribed. The inducer may be a chemical agent, such as a metabolite, growth regulator, herbicide, or phenolic compound, or physiological stress directly imposed on the plant, such as cold, heat, salt, toxins, or by the action of a pathogen or disease agent, such as a virus or fungus. A plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, washing, heating, or by exposure to the operative pathogen. An example of an inducible promoter suitable for use in the present invention is a glucocorticoid-inducible promoter ((Schena et al., "A Steroid-Inducible Gene Expression System for Plant Cells," Proc. Natl. Acad. Sci. 88:10421-5 (1991), which is incorporated herein by reference in its entirety). Expression of the protein encoded by the transgene is induced in the transformed plants when the transgenic plants are brought into contact with nanomolar concentrations of a glucocorticoid, or by contact with dexamethasone, a glucocorticoid analog (Schena et al., "A Steroid-Inducible Gene Expression System for Plant Cells," Proc. Natl. Acad. Sci. USA 88:10421-5 (1991); Aoyama et al., "A Glucocorticoid-Mediated Transcriptional Induction System in Transgenic Plants," Plant J. 11:605-612 (1997), and McNellis et al., "Glucocorticoid-Inducible Expression of a Bacterial Avirulence Gene in Transgenic Arabidopsis Induces Hypersensitive Cell Death," Plant J. 14(2):247-57 (1998), which are herein incorporated by reference in their entirety). In addition, inducible promoters include promoters that function in a tissue-specific manner to regulate the gene of interest within selected tissues of the plant. Examples of such tissue-specific or regulated promoters include seed, flower, fruit, or root specific promoters as are well known in the art (U.S. Patent No. 5,750,385 issued to Shewmaker et al., which is herein incorporated by reference in its entirety). In a preferred embodiment of the present invention, a heterologous promoter is linked to the constructor nucleic acid, where heterologous promoter is defined as a promoter to which the constructor nucleic acid is not naturally linked.

[040]The expression system of the present invention may also include an operable 3' regulatory region, selected from those that are capable of providing for the correct transcription termination and polyadenylation of the mRNA for expression in the host cell of choice, operably linked to a DNA molecule encoding a protein of choice.

[041]The vector of choice, promoter and an appropriate 3' regulatory region can be ligated together to produce the DNA construct of the present invention using well-known molecular cloning techniques as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989) and Current Protocols in Molecular Biology, New York, N.Y: John Wiley & Sons, (1989), which is hereby incorporated by reference in its entirety.

[042] Expression efficiency can be improved by the inclusion of appropriate transcription or translation enhancer elements (e.g., elements disclosed in Bittner et al., Methods in Enzymol. 153:516(1987), which is incorporated herein by reference in its entirety). Additionally, the genetic sequence can be modified for optimal codon usage in the appropriate expression system or, alternatively, the expression host can be modified to express specific tRNA molecules to facilitate expression of the desired gene.

[043] In addition, the recombinant expression vector may contain additional nucleotide sequences. For example, the recombinant expression vector may encode a selectable marker gene to identify host cells that have taken up the vector. In addition, to facilitate secretion of the protein from a host cell, the recombinant expression vector may encode a signal sequence linked to the amino terminus of the protein, such that upon expression, the protein is synthesized with the signal sequence fused to its amino terminus. This signal sequence directs the protein into the secretory pathway of the cell and is then normally cleaved, resulting in the release of the protein without the signal sequence from the host cell. The use of a signal sequence to facilitate secretion of protein or peptides from mammalian host cells is well known in the art.

[044] Once an expression system containing a polynucleotide according to the present invention has been prepared, it is ready for incorporation into a host cell. Basically, this method can be carried out by transforming a host cell with the expression system of the present invention under conditions effective for producing transcription of the DNA molecule in the host cell. The recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the host cell using standard cloning procedures known in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, New York (1989), which is incorporated herein by reference in its entirety. Suitable host cells include, but are not limited to, bacteria, viruses, yeast, mammalian, insect, plant cells, and so on. The transformation methods can result in transient or stable expression of the nucleic acid under control of the promoter. In one embodiment, a nucleic acid construct of the present invention is stably inserted into the recombinant genome of the plant cell as a result of transformation, although transient expression may serve an important purpose, particularly when the plant under investigation is slow growing.

[045]Plant tissue suitable for transformation includes leaf tissue, root tissue, meristems, zygotic and somatic embryos, callus, protoplasts, buds, pollen, embryos, anthers, and so on. The transformation media chosen consist of those most suitable for the tissue to be transformed.

[046] Transient expression in plant tissue is often achieved by particle bombardment (Klein et al., "High-Velocity Microprojectiles for Delivering Nucleic Acids Into Living Cells," Nature 327:70-73 (1987), which is hereby incorporated by reference in its entirety). In the present method, tungsten or gold microparticles (1 to 2 μm in diameter) are coated with the DNA of interest and then bombarded into the tissue using high-pressure gas. In this way, it is possible to release the foreign DNA into the nucleus and obtain temporal expression of the gene under the current tissue conditions. Biologically active particles (e.g., dried bacterial cells containing the heterologous vector DNA) can also be propelled into plant cells. Other variations of particle bombardment, now known or later developed, can also be used.

[047] A suitable method of stably introducing the nucleic acid construct into plant cells is to infect a plant cell with Agrobacterium tumefaciens or Agrobacterium rhizogenes previously transformed with the nucleic acid construct. As described above, the Ti (or RJ) plasmid of Agrobacterium allows for the highly successful transfer of a foreign nucleic acid molecule into plant cells. Another approach to transforming plant cells with a gene that communicates resistance to pathogens is particle bombardment (also known as biolistic transformation) of the host cell, as disclosed in United States Patent Nos. 4,945,050, 5,036,006 and 5,100,792, all to Sanford et al. and in Emerschad et al., "Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera)," Plant Cell Reports 14:6-12 (1995), which are incorporated herein by reference in their entirety. Still another method of introduction is the fusion of protoplasts with other entities, minicells, cells, lysosomes, or other fusible lipid-smoothed bodies (Fraley et al., Proc. Natl. Acad. Sci. USA 79:1859-63 (1982), which is incorporated herein by reference in its entirety). The nucleic acid molecule can also be introduced into plant cells by electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985), which is incorporated herein by reference in its entirety). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. High-power electrical pulses reversibly permeabilize biomembranes allowing introduction of the plasmid. Electroporated plant protoplasts reform the cell wall, divide and regenerate. The exact method of transformation is not critical to the practice of the present invention. Any method resulting in efficient transformation of the host cell of choice is suitable for practicing the present invention.

[048] After transformation, the transformed plant cells must be regenerated. Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); Vasil I.R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. Ill (1986), and Fitch et al., "Somatic Embryogenesis and Plant Regeneration from Immature Zygotic Embryos of Papaya (Carica papaya Z-.)," Plant Cell Rep. 9:320 (1990), which are incorporated herein by reference in their entirety.

[049]Media for regeneration varies from plant species to plant species, but generally a suspension of transformed protoplasts or a petri dish containing explants is the first step. Callus tissue is formed and shoots can be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. Culture media will usually contain various amino acids and hormones, such as auxins and cytokinins. Efficient regeneration will depend on the medium, the genotype and the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.

[050] Preferably, the transformed cells are first identified using a selection marker simultaneously introduced into the host cells along with the nucleic acid construct of the present invention. Suitable selection markers include, without limitation, markers encoding antibiotic resistance, such as the nptll gene that confers kanamycin resistance (Fraley et al., Proc. Natl. Acad. Sci. USA 80:4803-4807 (1983), which is incorporated herein by reference in its entirety) and genes conferring resistance to gentamicin, G418, hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and so on. The cells or tissues are cultured in a selection medium containing the appropriate antibiotic, whereby generally only those transformants express increasing resistance to the antibiotic marker. Other types of markers are also suitable for inclusion in the expression cassette of the present invention. For example, a gene that encodes herbicide tolerance, such as sulfonylurea tolerance, is useful, or the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2:1099-1104 (1983), which is incorporated herein by reference in its entirety). Similarly, "reporter genes," which encode enzymes that provide for the production of an identifiable compound, are suitable. The most widely used reporter gene for gene fusion experiments has been uidA, an Escherichia coli gene that encodes the β-glucuronidase protein, also known as GUS (Jefferson et al., "GUS Fusions: β Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher Plants," EMBO J. 6:3901-3907 (1987), which is incorporated herein by reference in its entirety). Similarly, enzymes that provide for the production of a compound identifiable by luminescence, such as luciferase, are useful. The selection marker employed will depend on the target species; for certain target species, different antibiotic, herbicide, or biosynthetic selection markers are preferred.

[051]Plant cells and tissues selected by means of an inhibitory agent or other selection marker are then tested for viral gene acquisition by Southern blot hybridization analysis using a probe specific for the viral genes contained in the cassette provided for transformation (Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor, New York: Cold Spring Harbor Press (1989), which is hereby incorporated by reference in its entirety).

[052] After the fusion gene containing a nucleic acid construct of the present invention is stably incorporated into transgenic plants, the transgene can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending on the species to be crossed. Once transgenic plants of this type are produced, the plants themselves can be grown as per conventional procedure so that the nucleic acid construct is present in the resulting plants. Alternatively, transgenic seeds are recovered from the transgenic plants. These seeds can then be planted in soil by conventional procedures and grown to produce transgenic plants.

[053] The present invention may be used in conjunction with a wide variety of plants or their seeds. Suitable plants include dicotyledons and monovotyledons. Useful crop plants may include: alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, Brussels sprouts, beet, parsnip, turnip, cauliflower, broccoli, rutabaga, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, papaya, sugar cane, and coffee.

[054] A further aspect of the present invention is directed to a method of degrading mannans and polysaccharides in plant material. This method involves providing plant material and contacting the plant material with the β-mannanase protein of the present invention under conditions effective for degrading mannans and polysaccharides in the plant material.

[055] In a preferred embodiment, the plant material that is contacted with the β-mannanase protein of the present invention is coffee beans, although other plant materials where degradation of mannans and polysaccharides is desired may also be contacted.

[056]It may also be desirable, according to this method of the present invention, to recover soluble sugar from degraded plant material, particularly that resulting from the step of contacting the plant material with the β-mannanase protein.

[057] These aspects of the present invention are also illustrated by the examples below.

EXAMPLES.

[058]The following examples are provided to illustrate embodiments of the present invention, but they are in no way intended to limit its scope.

Example 1. Cloning of β-mannanase from the midgut of the coffee berry borer (Hypothenemus hampei). Preparation of the Exhibition.

[059]Coffee seeds are infected with adult Hypothenemus hampei (Coleoptera: Scolytidae) insects as described in Rubio et al., “Morfologia Del Sistema Digestivo de Hypothenemus hampei (Ferrari)”, Cenicafe (Colombia) 58:66-74(2007), which is incorporated herein by reference in its entirety. Briefly, infected coffee beans are dissected with an incision using a stereomicroscope and larvae are collected in a Petri dish. Each larva is stored at 4 °C for at least 10 min to reduce physical activity and then placed on a glass slide with a drop of sterile distilled water and dissected under a Zeiss Estemi 2000 stereomicroscope using small forceps and 0.15 mm puncture needles. To isolate midgut tissue, each larva was dissected with an incision at the prothorax and midthorax, then a large incision along the length of the larvae to expose the entire alimentary canal. Finally, after removal of the fatty tissue, the midgut region was dissected and immediately deposited in a pre-chilled microcentrifuge tube with 100 μl of sterile distilled water containing 0.1% RNA from Later® reagent. All midgut tissues and dissected tissues were stored at -80 °C until protein extraction.

Construction of YSST Coffee Berry Borer Library.

[060]RNA was extracted from midgut tissues and stored at -80 °C. Polyadenylated RNA was isolated from total RNA using the Oligotex mRNA Midi Kit (Qi-agen, Valencia, CA) according to the manufacturer's instructions. First-strand cDNA were synthesized from mRNA using primer N6-NotI:5'-GAGAGAGAGAGAGAGAGAACCGCGCGGCCGCCNNNNNN-S' (SEQ ID NO: 4) with a cDNA Synthesis Kit (Stratagene). After second-strand synthesis, cDNAs were prepared by unidirectional cloning by ligation with EcoRI adapters according to the manufacturer's instructions, followed by digestion with N6-NotI. The cDNAs were then fractionated on 1% agarose gel electrophoresis, and those within the predicted size range of 300 to 1,000 bp were excised from the gel and purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA). The fragments were ligated to an equipartite mixture of the three vectors, PYSSTO, pYSST1, and pYSST2, digested with EcoR1 and NotI. TOP10F' electrocompetent cells (Invitro, Carsbad, CA) were transformed with approximately 1 μg of the resulting YSST library by electroporation (Micropulser Electroporator, Bio-Rad, Hercules, CA) and spread onto 10 to 15 large LB plates. Plasmid DNA was isolated from a pool of the resulting transformants using the Perfectprep Plasmid Midi kit (Eppendorf). Fifty microgammas from the YSST library were transformed into yeast ( Saccharomyces cerevisiae ) strain DBYα2445 (MATα, suc2Δ-9, lys2-801, ura3-52, ade2-101) using the YEASTMAKER Yeast Transformation System (BD Biosciences, San Jose, CA). Transformants were streaked onto YP sucrose plates (1% yeast extract, 2% peptone, 2% sucrose, 2% agar, pH 6.5), incubated at 30 °C for 4 to 9 days, and visible colonies were streaked onto the sucrose plate followed by incubation at 30 °C for 2 to 3 days. Plasmids are isolated from visible colonies as described in Hoffmann and Winston, "A Ten-Minute DNA Preparation from Yeast Efficiently Releases Autonomous Plasmids for Transformation of Escherichia coli" Gene 57:267-272 (1987), which is incorporated herein by reference in its entirety, transformed into XL1-blue electrocompetent E. coli, and purified using a Qiaprep kit (Qiagen). Plasmid inserts are sequenced using a primer equivalent to the ADH1 promoter of pYSSTO, pYSST1, pYSST2 (5'-TCCTCGTC ATTGTTCTCGTTCC-3') (SEQ ID NO: 5) at the Bio Resource Center, Cornell University, Ithaca, NY (http://www.brc.cornell.edu).

RACE amplification.

[061] An oligonucleotide primer derived from the DNA sequence equivalent to the predicted signal sequences of the isolated mannanase clone YSST was used in the 3'RACE with the primer as an adapter primer. To obtain the full-length cDNA sequence, 'landing' PCR was performed using a program with 35 cycles of 94 °C for 1 min, 63 °C for 1 min, 72 °C for 2.5 min with the annealing temperature reduced by 1 °C every second cycle to 60 °C, followed by the final extension at 72 °C for 10 min where the PCR products were subcloned into the pGEM-T Easy vector (Promega, Madison, Wisconsin). DNA sequences are determined as described above. H. hampei β-mannanase is an endoglucanase (endo-β-1,4-D-glucanase, EC 3.2.1.4). It is a single polypeptide chain of 320 amino acids, with a predicted molecular mass of 35.62 kDa and a theoretical pI of 4.72, calculated from the amino acid composition.

Example 2. Heterologous expression of β-mannanase in Spodoptera frugiperta (Sf9) cells. Construction of Fusion Protein Expression Plasmid.

[062] For the construction of the C-terminal na-HIS tagged mannanase expression plasmid, the cDNAs are reamplified by PCR using a mannanase cDNA in the Easy pGEM-T vector (Promega, Madison, Wisconsin) as a template with the following primers: 5'-CACCATGGAACCTTTTGTGGTC-3' (SEQ ID NO: 6) and 5'-GACAGGGATGAAGCAGATCTGG-3'' (SEQ ID NO: 7).

[063]The underlined portion of SEQ ID NO: 6 was introduced for directional cloning of TOPO. The last reverse primer requires the stop codon of the native β-mannanase cDNA. The resulting cDNA was cloned into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA) and named pENTR/β-Mann.

Baculovirus DNA Recombination and Transfection.

[064]Baculovirus construction and protein expression in Sf9 cells are performed according to the BaculoDirect Baculovirus Expression System protocol from Invitrogen (Carlsbad, CA). Spodoptera frugiperda Sf9 cells were transfected with recombinant bacmid DNA for production of baculovirus particles. Cells are grown at 27 °C in SF900-II medium (Life Technologies) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. For transfection, 9x105 cells were plated in 35 mm tissue culture flasks and incubated for 1 h in 2 ml Sf900-II SFM (Life Technologies) without antibiotics to allow cell adhesion to the plate. The medium is then changed to 1 ml of antibiotic-free Sf900-II serum containing recombinant bacmid DNA (5 μl of a standard plasmid DNA mini-prep) that had been preincubated for 30 min at room temperature with CellFectin (6 μl) (Life Technologies). The cells were incubated with the liposome-DNA complex at 27 °C for 5 h. The transfection medium was removed and 2 ml of SF900-II medium containing antibiotics was added. The pENT®/Man plasmid was transfected into Sf9 cells and non-recombinant bacmid (Bd) DNA and pENT®/CAT were used as negative and positive controls, respectively. The transfected cells were incubated at 27 °C for 72 h which allowed the production and release of baculovirus into the culture medium. The culture medium from each transfection was collected, clarified (500 g for 5 min), and stored at 4°C as a master virus stock. Transfection efficiency, recombinant baculovirus (Bv-Man), and nonrecombinant baculovirus (Bv) production were monitored by visualizing the cytopathic exposure effect of transfected cells within 48 h after subculture under a phase-contrast microscope and analyzing the presence of baculovirus DNA by PCR analysis. At this point, the presence of baculovirus in 50 μl of infected culture supernatant is pelleted at 12,000 g for 10 min in a microcentrifuge tube and a volume (25 μl) of proteinase K buffer (10 Tris-HCl, pH 7.8; 5 mM EDTA; 0.5% SDS) containing 50 μg/ml proteinase K (Sambrook et al, 1989) is added to the pellet to digest viral protein for 1 h at 56°C. An additional heating at 95°C for 20 min is included to inactivate the enzyme before proceeding to the PCR step. Amplification of viral DNA is performed using 2 μl of this DNA preparation as the template under the same conditions and primers described above. The cells were selected with ganciclovir for 120 hours and the resulting viral stock was amplified twofold by infection of Sf9 cells.

Amplification of Baculovirus Stocks.

[065] For amplification of baculovirus master stocks, 1x106 Sf9 cells are plated in a 25 cm2 flask and incubated for 1 h with 10 μl of the baculovirus master stock in 1 ml of SF900-II medium containing antibiotics (corresponding to an MOI of 0.01 to 0.1). After this period, the medium was completed to 4.5 ml and the infected cells were incubated for 48 h at 27 °C. The culture medium was collected, clarified (500 g for 5 min) and stored at 4 °C as viral stocks for recombinant protein production.

Scale-up of Recombinant β-mannanase Production.

[066]S. frugiperta cells are plated in a 225 cm2 dish with 50 ml of culture medium and incubated as above. Cells are transfected in log phase using 500 μl of a P3 viral stock (titer 3.44 x 106 pfu/ml). After 96 hours, the culture medium was harvested, clarified (500 g for 5 min) and the recombinant β-mannanase used was purified from the MagneHis ® System (Promega, Madison, Wisconsin). This resulted in the production of 2.4 mg of purified β-mannanase per 100 ml of culture.

Example 3. Purification of recombinant β-mannanase.

[067] MagneHis ® Ni-particle pull-down assays (Promega, Madison, Wisconsin) are performed according to the manufacturer's protocol. Briefly, 10 ml of culture medium after cell removal was spiked with 300 μl of MagneHis ® Ni-particles and incubated at room temperature for 2 min with gentle shaking. After incubation, the tube was placed on a magnetic rack for 30 s to allow the MagneHis ® Ni-particles to be captured by the magnet and the supernatant was removed. The MagneHis ® Ni-particles were washed three times with binding/wash buffer. Pure 6xHis-tagged β-mannanase protein was subjected to SDS/PAGE (Figure 1) and functional assay (Figure 2).

Example 4. Determination of Enzyme Activity Using β - Galactomannans from Carob Tree Seeds.

[068]Cross-linked Azurine-Galactomannan was prepared by dyeing and cross-linked galactomannan polysaccharide extracted from carob seed flour (AZCL-® galactomannan; Megazyme International Ireland Ltd.). The Carob tree, Ceratonia siliqua, is an evergreen shrub or tree, native to the Mediterranean region, cultivated for its edible seed pods. This substrate is insoluble in buffered solutions but rapidly hydrates to form gel particles that are readily and rapidly hydrolyzed by specific soluble endohydrolases released from dye-labeled fragments according to Marraccini et al., "Molecular and Biochemical Characterization of ENDO-β-MANNANASES from Germinating Coffee (Coffea arabica) Grains," Planta 213:296308 (2001), which is incorporated herein by reference in its entirety. A 20 μl aliquot of 6xHis-tagged recombinant β-mannanase protein mixed in the substrate solution [1% (w/v) AZCL-galactomannan® in 0.2 M acetic buffer (pH 5.0)] was incubated at 37 °C with gentle shaking. The 200 μL aliquots were removed after 30 min and heated at 100 °C for 5 min to stop the reaction. Each aliquot was centrifuged at 13000 rpm for 5 min and the absorbance was measured in À ~ s nm (Figure 2).

Example 5. Determination of Enzyme Activity Using β - Galactomannan from Coffee Beans Purification of β -Galactomannan from Coffee.

[069] A sequential fractionation procedure based on a delignification treatment, an acid wash and subsequent alkali extraction (as in Bradbury et al., "Chemical Structures of Green Coffee Bean Polysaccharides," J. Agric. Food Chem. 38:389-392 (1990), which is incorporated herein by reference in its entirety) was used to isolate pure β-mannan from green coffee beans. Green Coffee arabica coffee beans are Soxhlet extracted with chloroform/methanol (2:1) and petroleum ether (5H) for removal of lipids and with aqueous ethanol (95%, overnight) for removal of low molecular weight carbohydrate. The defatted beans are hot water extracted and then delignified with weakly acidic sodium chloride solution according to the method of Wolfrom and Patin, "Carbohydrates of the Coffee Bean. IV. An Arabinogalactan," J. Org. Chem. 30:4060-4063 (1965), which is hereby incorporated by reference in its entirety, to provide a white holocellulose product. The major portion of the arabinogalactan polymer is solubilized, in a partially hydrolyzed form, by washing with dilute hydrochloric acid (1%, 80°C). The mannans are then isolated into discrete fractions by extraction (overnight, 4°C) with 2.5 and 10% sodium hydroxide solutions. Addition of ethanol to the 2.5% NaOH extracts yields a precipitate containing arabinogalactan and mannan. Neutralization of the 10% NaOH extracts led to the rapid formation of a white precipitate, which was removed by filtration after stirring was allowed to stand overnight at 4 °C. A fraction was also obtained by addition of ethanol to the filtrate. The precipitate was washed with ethanol and diethyl ether before drying. The mannan substrate used in the present work is the fraction precipitated by neutralization of the 10% NaOH extracts, which contained 94% mannan by weight.

Effect of pH and Temperature on the Activity of Recombinant B-Mannanase from H. hampei.

[070] To test the enzyme activity against coffee galactomannans, the α-mannanase activity was determined by the dinitrosalicylic acid assay of Bernfeld, P. In: Collowick S. P. and Kaplan N. O. (eds.), Methods in Enzymology, Vol. I Aca-demic Press, New York, pp. 149-158 (1955), which is hereby incorporated by reference in its entirety. The optimum pH of the enzyme activity against coffee galactomannan was determined at different pH values ranging from 4.0 to 11.0. The buffer is 200 mM sodium acetate and 100 mM sodium chloride at 37 °C. The highest enzyme activity was observed at pH 6.0 (Figure 3). The effect of temperature on the activity of recombinant β-mannanase against coffee galactomannan was examined in the temperature range from 15 °C to 70 °C using the same buffer at pH 6.0 (Figure 4). The enzyme showed maximum activity around 50 °C. The enzyme activity decreased sharply with further increases in temperature.

[071] Although the invention has been described in detail for purposes of illustration, it is understood that such detail is only so that the objectives and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims (6)

1. Isolated expression system CHARACTERIZED by the fact that it comprises a DNA construct consisting of the sequence defined by SEQ ID NO: 2 operably linked to heterologous transcription promoter and terminator sequences. 2. Isolated host cell CHARACTERIZED by the fact that it contains a polynucleotide defined by SEQ ID NO: 2, in which the isolated host cell is selected from bacterial cells. 3. A method of recombinantly producing a β-mannanase protein, said method comprising: providing the isolated host cell as defined in claim 2; culturing the isolated host cell under conditions effective for the host cell to express the β-mannanase protein; and recovering the β-mannanase protein. 4. A method of degrading mannans and polysaccharides in a plant material, said method comprising: providing the plant material; and contacting the plant material with a β-mannanase protein having an amino acid sequence defined by SEQ ID NO: 1 under conditions effective to degrade the mannans and polysaccharides in the plant material. 5. The method of claim 4, wherein the plant material is coffee beans. 6. The method of claim 4, further comprising recovering soluble sugars from the degraded plant material resulting from the plant material with the β-mannanase protein.