Abstract:
The object of the invention is a novel plasmidic gene encoding an amylolytic enzyme which after introduction into a microbial strain, favorably bacterial, especially  Lactococcus lactis , enables the production of the encoded amylolytic enzyme, the ways of acquiring this gene and the industrial application of the enzyme it encodes.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     Lactic acid bacteria are capable of utilizing many different sugars as a source of carbon and energy (De Vos W. 1996. Antonie van Leeuwenhoek 70: 223). In a significant majority these sugars are monosaccharides or disaccharides. The ability of lactic acid bacteria to catabolize carbohydrates is widely used in biotechnological processes linked with food production (Libudzisz Z., Walczak P. and Bardowski J. (ed.), 1998. Lactic Acid Bacteria—classification, metabolism, genetics, application. (In polish) Monographies, ódź; Aleksandrzak T., Kowalczyk M., Kok J., Bardowski J. 2000, Food Biotechnol. 17: 61, Elsevier Science B. V., Amsterdam). One of these processes is the production of fermented milk products. The main sugar in milk is lactose, which catabolism is a feature rather commonly present among lactic acid bacteria (Bardowski J. 1995. Przeglad Mleczarski 11: 315; Van Rooijen R. J. 1992. Lactose catabolism in  Lactococcus lactis.  Ph.D thesis, Wageningen, The Netherlands).  
         [0002]     Yet another natural source, besides milk, of food production are plants. These biotechnological processes are frequently conducted with the use of lactic acid bacteria. However, in plant material, differently than in milk, polysaccharides such as cellobiose or starch are present. Thus, in the microbial conversion of plant material these microbials are incorporated which have either cellulolytic or amylolytic abilities. Lactic acid bacteria lack the ability to degrade cellulose.  
         [0003]     However, the ability to degrade starch is, in this group of bacteria, a feature limited basically to a few strains of the  Lactobacillus  genus (cit. after Giraud E., A. Chapailler and M. Raimbault. 1994. Appl. Environ. Microbiol. 60: 4319). Some of these bacteria, belonging to the  L. plantarum, L. amylophilus,  or  L. amylovorus  species produce α-amylase (Fitzsimons A. and O&#39;Connell. 1994. FEMS Microbiol. Letts. 116:137; Pompeyo C C., Gomez M S., Gasparian S., J. Morlon-Guyot 1993. Appl. Microbiol. Biotechnol. 40: 266).  
         [0004]     It has been determined that besides strains from  Lactobacillus  genus, some strains of  Lactococcus  genus, naturally existing in the environment, also demonstrate this since strains which degrade starch were found among  Lactococcus  strains isolated from natural habitats (Domań M., E. Czerniec, Z. Targoński and J. Bardowski. (2000), Food Biotechnology 17:67, Elsevier Science B.V., Amsterdam).  
         [0005]     Unexpectedly, it has been discovered that also other strains, especially from the  Lactococcus lactis  species, after introducing a specific gene structure according to the invention, are capable of producing a novel amylolytic enzyme which is encoded by this gene and which is secreted into the environment.  
       SUMMARY OF THE INVENTION  
       [0006]     This invention is directed to a novel plasmidic gene encoding an amylolytic enzyme which after introduction into a microbial strain, favorably bacterial, especially  Lactococcus lactis , enables the production of the encoded amylolytic enzyme, as well as ways of obtaining this gene and the industrial application of the enzyme it encodes. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0007]     The novel plasmidic gene encoding the novel amylolytic enzyme secreted into the environment, according to the invention has the following nucleotide sequence (top strand: SEQ. ID NO. 1 and bottom strand: SEQ. ID NO. 2):  
                                   5′                       ATGAAAAAAACAAAATTAAGAATTTGTCTTTTCCCAATCGTACTGCTTATCAGTTTTACA          1   ---+---------+---------+---------+---------+---------+------     60           TACTTTTTTTGTTTTAATTCTTAAACAGAAAAGGGTTAGCATGACGAATAGTCAAAATGT                   TTGCTATCCAATGGAACTGGTAAACAGATTGTTTTTGCTACATCTTCATCAGAATATGCT         61   ---+---------+---------+---------+---------+---------+------    120           AACGATAGGTTACCTTGACCATTTGTCTAACAAAAACGATGTAGAAGTAGTCTTATACGA                   ACACAAGCTCTGTCTGACGAGAAAAATGCCACACAAAACAATGATTTTACATCTTTTGAT        121   ---+---------+---------+---------+---------+---------+------    180           TGTGTTCGAGACAGACTGCTCTTTTTACGGTGTGTTTTGTTACTAAAATGTAGAAAACTA                   ATAAATGGGCCTATGAAGGGACAGATTTGGGATTTAACTATTCAACAACCAGTACTACT        181   ---+---------+---------+---------+---------+---------+------    240           TATTTTACCCGGATACTTCCCTGTCTAAACCCTAAATTGATAAGTTGTTGGTCATGATGA                   TTCAAAATCTGGTCACCAACTGCGACGAGCGTGCAGCTGATAAGTTATGGGACGAATACC        241   ---+---------+---------+---------+---------+---------+------    300           AAGTTTTAGACCAGTGGTTCACGCTGCTCGCACGTCGACTATTCAATACCCTGCTTATGG                   AATCCGACAGCGGCACAGGTCTCAGCGAAAGCAATGACACGCGGGACATCAGCGACACCA        301   ---+---------+---------+---------+---------+---------+------    360           TTAGGCTGTCGCCGTGTCCAGAGTCGCTTTCGTTACTGTGCGCCCTGTAGTCGCTGTGGT                   ACTAATCACGCAACCAATACGATTGGGGTGTGGACTTTGACCGTTCCTGGCAATCAAAAT        361   ---+---------+---------+---------+---------+---------+------    420           TGATTAGTGCGTTGGTTATGCTAACCCCACACCTGAAACTGGCAAGGACCGTTAGTTTTA                   GGCATGGTTTATGCTTACAAACTAACTTTTGCTGACGGAACCGTTAGTGATTATGCTGGG        421   ---+---------+---------+---------+---------+---------+------    480           CCGTACCAAATACCAATGTTTGATTGAAAACGACTGCCTTGGCAATCACTAATACGACCC                   TCAACTTATGGAACACTTTCTACAAGTTCTGTCAGCAATACAACCAATGATCCTTATTCT        481   ---+---------+---------+---------+---------+---------+------    540           AGTTGAATACCTTGTGAAAGATGTTCAAGACAGTCGTTATGTTGGTTACTAGGAATAAGA                   ATTGCGACGACACAAGGTGGAAATCGTTCAGTTGTCGAATCGTCTGCAAACCTCGCGTCA        541   ---+---------+---------+---------+---------+---------+------    600           TAACGCTGCTGTGTTCCACCTTTAGCAAGTCAACAGCTTAGCAGACGTTTGGAGCGCAGT                   AATCTGGCACTTGCGCAAGGCAAATCTGCAACTTGGCGGGTAGCAAGTCCAACACAAGCC        601   ---+---------+---------+---------+---------+---------+------    660           TTAGACCGTGAACGCGTTCCGTTTAGACGTTGAACCCCCCATCGTTCAGCTTGTGTTCGG                   ATCGTTGACGAACTACATATTCGTGATTTTACAAGCTCATCAACATCTGGTGTTTCGGCT        661   ---+---------+---------+---------+---------+---------+------    720           TAGCAACTGCTTGATGTATAAGCACTAAAATGTTCGAGTAGTTGTAGACCACAAAGCCGA                   GGTAATCGTGGTAAGTTTCTCGGAGTGATTCAGTCAGGAACAACTGATCCAAATACAGGT        721   ---+---------+---------+---------+---------+---------+------    780           CCATTAGCACCATTCAAAGAGCCTCACTAAGTCAGTCCTTGTTGACTAGGTTTATGTCCA                   ACAGCGACTGGACTTGATTATCTGAAAAACGAGGGTTTTAACTACATTCAACTTCATGCC        781   ---+---------+---------+---------+---------+---------+------    840           TGTCGCTGACCTGAACTAATAGACTTTTTGCTCCCAAAATTGATGTAAGTTGAAGTACGG                   AGCCAGTCAGTATGCGTCGGTCAACGAAGCCGGAACCGTACTACTGCGCAACCGAATAAC        841   ---+---------+---------+---------+---------+---------+------    900           TCGGTCAGTCATACGCAGCCAGTTGCTTCGCCCTTGGCATGATGACGCGTTGGCTTATTG                   TTCAACTGGGGATATGACCCACAAATGAAATGGTACCAGAGCGAATATGCCAGTAATTCT        901   ---+---------+---------+---------+---------+---------+------    960           AAGTTGACCCCTATACTGGGTGTTTACTTTACCATGGTCTCGCTTATACGGTCATTAAGA                   GTCAATCCTGTCACACGTATTAATGAAATGAAAGAAATGGTACAAGGCTTGCATACAAAT        961   ---+---------+---------+---------+---------+---------+------   1020           CAGTTAGGACAGTGTGCATAATTACTTTACTTTCTTTACCATGTTCCGAACGTATGTTTA                   GGGATTAGCGTGGTAATGGATATGGTTCTCAATCACGTCTATAGTCAGTCAGCATCCGGT       1021   ---+---------+---------+---------+---------+---------+------   1080           CCCTAATCGCACCATTACCTATACCAAGAGTTAGTGCAGATATCAGTCAGTCGTAGGCGA                   TTTGAAAAAGCCGAGCCGGGCTATTATTTCCGCAAAAATACGCAATCTGGGTGTGGCAAT       1081   ---+---------+---------+---------+---------+---------+------   1140           AAACTTTTTCGGCTCGGCCCGATAATAAAGGCGTTTTTATGCGTTAGACCCACACCGTTA                   GATACGGCGAGCAACCACGAAATGTTTGGTAAATACATTATTGACTCTGTCACTTACTGG       1141   ---+---------+---------+---------+---------+---------+------   1200           CTATGCCGCTCGTTGGTGCTTTACAAACCATTTATGTAATAACTGAGACAGTGAATGACC                   GCGAAAAATTATGATATTGACGGCTTCCGTTTTGATGAAATGACACTTTTAGACAGCACG       1201   ---+---------+---------+---------+---------+---------+------   1260           CGCTTTTTAATACTATAACTGCCGAAGGCAAAACTACTTTACTGTGAAAATCTGTCGTGC                   ACTATGAATAAACTGCGTGCCGCACTGACAGCACTTGACCCACATATTATCATGTATGGC       1261   ---+---------+---------+---------+---------+---------+------   1320           TGATACTTATTTGACGCACGGCGTGACTGTCGTGAACTGGGTGTATAATAGTACATACCG                   GAAGGTTGGGGCGATTCTAATGCAAATAATATTCCTGAAACGTCTATCAATAATTACAAA       1321   ---+---------+---------+---------+---------+---------+------   1380           CTTCCAACCCCGCTAAGATTACGTTTATTATAAGGACTTTGCAGATAGTTATTAATGTTT                   AATGTTCCTGGTATTGGTTTTTTCAATCCTGGTGAACGTGACGCCATCAGTAATAATGGA       1381   ---+---------+---------+---------+---------+---------+------   1440           TTACAAGGACCATAACCAAAAAAGTTAGGACCACTTGCACTGCGGTAGTCATTATTACCT                   GGTTCTGCAGGTGGATTCGCAGCAGGAAATACGGCAAGTACTATAACGGTCGCAGGAGCA       1441   ---+---------+---------+---------+---------+---------+------   1500           CCAAGACGTCCACCTAAGCGTCGTCCTTTATGCCGTTCATGATATTGCCAGCGTCCTCGT                   CTTTTGGCTTCTGCAGGTTGGAACGGAAATGGCACAGTGCAAGCTTTTTTGACGCCAAGC       1501   ---+---------+---------+---------+---------+---------+------   1560           GAAAACCGAAGACCTCCAACCTTGCCTTTACCGTGTCACGTTCGAAAAAACTGCGGTTCG                   CAGTCTATCAATTATGTTGAATGTCATGATAGCTTCACCTTAAATGACTCACTCTGGTCA       1561   ---+---------+---------+---------+---------+---------+------   1620           GTCAGATAGTTAATACAACTTACAGTACTATCGAAGTGGAATTTACTGAGTGAGACCAGT                   GCTGATCCGAATGATTCTGTGGCGACACATCAAGCGCGAGTGACGCTCGCAAATGCCACG       1621   ---+---------+---------+---------+---------+---------+------   1680           CGACTAGGCTTACTAAGACACCGCTGTGTAGTTCGCGCTCACTGCGAGCGTTTACGGTGC                   AATATTCTTGCA~ACGGTGTGACATTTATGGAAACAGGACAGGAGTTTGACCAGTCCAAA       1681   ---+---------+---------+---------+---------+---------+------   1740           TTATAAGAACGTTTGCCACACTGTAAATACCTTTGTCCTGTCCTCAAACTGGTCAGGTTT                   TTAGTCAATCCGTCAAATCTGACGCCGCTCTCCCCTACACAGACACAGGCTTATCAATCG       1741   ---+---------+---------+---------+---------+---------+------   1800           AATCAGTTAGGCAGTTTAGACTGCGGCGAGAGGGGATGTGTCTGTGTCCGAATAGTTAGC                   GGTAGTATGGAAAAACCTGCATGGTATCCTGCGTCATGGGATACTGCCAAAAATTCATAC       1801   ---+---------+---------+---------+---------+---------+------   1860           CCATCATACCTTTTTGGACGTACCATAGGACGCAGTACCCTATGACGGTTTTTAAGTATG                   AATGGACTTTTTGGCTTGGCTAGTAATGGTACTTACTATGGCAATTATTGGCCAGGAAGT       1861   ---+---------+---------+---------+---------+---------+------   1920           TTACCTGAAAAACCGAACCGATCATTACCATGAATGATACCGTTAATAACCGGTCCTTCA                   AATCTCTACACCCCAGTAGTTGCGGGGGATGTAGTCAATGCCATGAATTGGGATAATGTC       1921   ---+---------+---------+---------+---------+---------+------   1980           TTAGAGATGTGGGGTCATCAACGCCCCCTACATCAGTTACGGTACTTAACCCTATTACAG                   AAGGACAATCAAAATGCTGTTAATTTTATTGGAAATTTGATGAAATTTAAAAAATCCAAT       1981   ---+---------+---------+---------+---------+---------+------   2040           TTCCTGTTAGTTTTACGACAATTAAAATAACCTTTAAACTACTTTAAATTTTTTAGGTTA                   CCGCAATTTTGGCCTGATGATTATAGCAAACTTGCTTGGACTCCTACCAGTATAGGTGTA       2041   ---+---------+---------+---------+---------+---------+------   2100           GGCGTTAAAACCGGACTACTAATATCGTTTGAACGAACCTGAGGATGGTCATATCCACAT                   GAAAATGTCACAAATGCGTCAAACGGAGTGATCACAGAAGAATTAACATCGGGTGCGACC       2101   ---+---------+---------+---------+---------+---------+------   2160           CTTTTACAGTGTTTACGCAGTTTGCCTCACTAGTGTCTTCTTAATTGTAGCCCACGCTGG                   AAATATTTAGTCATATTGAATGCTAGCGGTAATTCGGTCAAAATTGGTCAAGGTGGTCAG       2161   ---+---------+---------+---------+---------+---------+------   2220           TTTATAAATCAGTATAACTTACGATCGCCATTAAGCCAGTTTTAACCAGTTCCACCAGTC                   TTTTATGGGGTGTCAAATTTGACAGGTAAAACTGTGATAATTTCTGACGATAGTAGCTTA       2221   ---+---------+---------+---------+---------+---------+------   2280           AAAATACCCCACAGTTTAAACTGTCCATTTTGACACTATTAAAGACTGCTATCATCGAAT                   ACAGCTAATCAAGTTTTGAATAGCTCTGTCACTCTGTCAAATTTGACAGTCACCGTTATC       2281   ---+---------+---------+---------+---------+---------+------   2340           TGTCGATTAGTTCAAAACTTATCGAGACAGTGAGACAGTTTAAACTGTCAGTGGCAATAG                   CAACTTTCTAAATAA       2341   ---+---------+- 2355             3′           GTTGAAAGATTTATT          
 
         [0008]     The novel gene according to the invention favorably has at its 5′ end an additional nucleotide sequence which together give the following nucleotide structure: (top strand: SEQ. ID NO. 3 and bottom strand: SEQ. ID NO. 4)  
                                       TTACGTTAGCTGGAGAATCCACCGTGTGGAGCCTCCGTCTCACGGTTGCTCCGTGTTACG                  1   ---------+---------+---------+---------+---------+---------+     60           AATGCAATCGACCTCTTAGGTGGCACACCTCGGAGGCAGAGTGCCAACGAGGCACAATGC                   GTAATTCCGCTGCTATAAAGTAATAACTAATAACAAATTAAATAGCAGTATAAAATTTTG         61   ---------+---------+---------+---------+---------+---------+    120           CATTAAGGCGACGATATTTCATTATTGATTATTGTTTAATTTATCGTCATATTTTAAAAC                   ATAATTTGAAGATTATTTTAATATTTACCTGAATAACACTTTAGAAAACTGAAATATCTA        121   ---------+---------+---------+---------+---------+---------+    180           TATTAAACTTCTAATAAAATTATAAATGGACTTATTGTGAAATCTTTTGACTTTATAGAT                   TAACTCCAGATCAATAGTGGAAATACGTACAATTTGATATAAGAGATTTTTGTATCAAAA        181   ---------+---------+---------+---------+---------+---------+    240           ATTGAGGTCTAGTTATCACCTTTATGCATGTTAAACTATATTCTCTAAAAACATAGTTTT                   AAAATTTTTTTAAACAAAATGCACTAA.AAGATTTTTTTGTGTTGCTGAGAGGTTGCAAAA        241   ---------+---------+---------+---------+---------+---------+    300           TTTTAAAAAAATTTGTTTTACGTGATTTTCTAAAAAAACACAACGACTCTCCAACGTTTT                   TGATACCGCAAGGTATATAATGGTTACGTAATTAGTGCAAGCGCTTGCATTTTTTGTGAA        301   ---------+---------+---------+---------+---------+---------+    360           ACTATGGCGTTCCATATATTACCAATGCATTAATCACGTTCGCGAACGTAAAAACACTT                   AAAAGTCCTTTTATTTATTTATCTTATTGCTTTGAAAGATATTGTTAAAAATAAATAAAA        361   ---------+---------+---------+---------+---------+---------+    420           TTTTCAGGAAAATAAATAAATAGAATAACGAAACTTTCTATAACAATTTTTATTTATTTT                   TCGTTATAAACTCAATATAATAAGGAGAGCTAAATAATGAAAAAAACAAAATTAAGAATT        421   ---------+---------+---------+---------+---------+---------+    480           AGCAATATTTGAGTTATATTATTCCTCTCGATTTATTACTTTTTTTGTTTTAATTCTTAA                   TGTCTTTTCCCAATCGTACTGCTTATCAGTTTTACATTGCTATCCAATGGAACTGGTAAA        481   ---------+---------+---------+---------+---------+---------+    540           ACAGAAAAGGGTTAGCATGACGAATAGTCAAAATGTAACGATAGGTTACCTTGACCATTT                   CAGATTGTTTTTGCTACATCTTCATCAGAATATGCTACACAAGCTCTGTCTGACGAGAAA        541   ---------+---------+---------+---------+---------+---------+    600           GTCTAACAAAAACGATGTAGAAGTAGTCTTATACGATGTGTTCGAGACAGACTGCTCTTT                   AATGCCACACAAAACAATGATTTTACATCTTTTGATATAAAATGGGCCTATGAAGGGACA        601   ---------+---------+---------+---------+---------+---------+    660           TTACGGTGTGTTTTGTTACTAAAATGTAGAAAACTATATTTTACCCGGATACTTCCCTGT                   GATTTGGGATTTAACTATTCAACAACCAGTACTACTTTCAAAATCTGGTCACCAACTGCG        661   ---------+---------+---------+---------+---------+---------+    720           CTAAACCCTAAATTGATAAGTTGTTGGTCATGATGkAAGTTTTAGACCAGTGGTTGACGC                   ACGAGCGTGCAGCTGATAAGTTATGGGACGAATACCAATCCGACAGCGGCACAGGTCTCA        721   ---------+---------+---------+---------+---------+---------+    780           TGCTCGCACGTCGACTATTCAATACCCTGCTTATGGTTAGGCTGTCGCCGTGTCCAGAGT                   GCGAAAGCAATGACACGCGGGACATCAGCGACACCAACTAATCACGCAACCAATACGATT        781   ---------+---------+---------+---------+---------+---------+    840           CGCTTTCGTTACTGTGCGCCCTGTAGTCGCTGTGGTTGATTAGTGCGTTGGTTATGCTAA                   GGGGTGTGGACTTTGACCGTTCCTGGCAATCAAAATGGCATGGTTTATGCTTACAAACTA        841   ---------+---------+---------+---------+---------+---------+    900           CCCCACACCTGAAACTGGCAAGGACCGTTAGTTTTACCGTACCAAATACGAATGTTTGAT                   ACTTTTGCTGACGGAACCGTTAGTGATTATGCTGGGTCAACTTATGGAACACTTTCTACA        901   ---------+---------+---------+---------+---------+---------+    960           TGAAAACGACTGCCTTGGCAATCACTAATACGACCCAGTTGAATACCTTGTGAAAGATGT                   AGTTCTGTCAGCAATACAACCAATGATCCTTATTCTATTGCGACGACACAAGGTGGAAAT        961   ---------+---------+---------+---------+---------+---------+   1020           TCAAGACAGTCGTTATGTTGGTTACTAGGAATAAGATAACGCTGCTGTGTTCCACCTTTA                   CGTTCAGTTGTCGAATCGTCTGCAAACCTCGCGTCAAATCTGGCACTTGCGCAAGGCAAA       1021   ---------+---------+---------+---------+---------+---------+   1080           GCAAGTCAACAGCTTAGCAGACGTTTGGAGCGCAGTTTAGACCGTGAACGCGTTCCGTTT                   TCTGCAACTTGGCGGGTAGCAAGTCCAACACAAGCCATCGTTGACGAACTACATATTCGT       1081   ---------+---------+---------+---------+---------+---------+   1140           AGACGTTGAACCGCCCATCGTTCAGGTTGTGTTCGGTAGCAACTGCTTGATGTATAAGCA                   GATTTTACAAGCTCATCAACATCTGGTGTTTCGGCTGGTAATCGTGGTAAGTTTCTCGGA       1141   ---------+---------+---------+---------+---------+---------+   1200           CTAAAATGTTCGAGTAGTTGTAGACCACAAAGCCGACCATTAGCACCATTCAAAGAGCCT                   GTGATTCAGTCAGGAACAACTGATCCAAATACAGGTACAGCGACTGGACTTGATTATCTG       1201   ---------+---------+---------+---------+---------+---------+   1260           CACTAAGTCAGTCCTTGTTGACTAGGTTTATGTCCATGTCGCTGACCTGAACTAATAGAC                   AAAAACGAGGGTTTTAACTACATTCAACTTCATGCCAGCCAGTCAGTATGCGTCGGTCAA       1261   ---------+---------+---------+---------+---------+---------+   1320           TTTTTGCTCCCAAAATTGATGTAAGTTGAAGTACGGTCGGTCAGTCATACGCAGCCAGTT                   CGAAGCGGGAACCGTACTACTGCGCAACCGAATAACTTCAACTGGGGATATGACCCACAA       1321   ---------+---------+---------+---------+---------+---------+   1380           GCTTCGCCCTTGGCATGATGACGCGTTGGCTTATTGAAGTTGACCCCTATACTGGGTGTT                   ATGAATGGTACCAGAGCGAATATGCCAGTAATTCTGTCAATCCTGTCACACGTATTAAT       1381   ---------+---------+---------+---------+---------+---------+   1440           TACTTTACCATGGTCTCGCTTATACGGTCATTAAGACAGTTAGGACAGTGTGCATAATTA                   GAAATGAAAGAAATGGTACAAGGCTTGCATACAAATGGGATTAGCGTGGTAATGGATATG       1441   ---------+---------+---------+---------+---------+---------+   1500           CTTTACTTTCTTTACCATGTTCCGAACGTATGTTTACCCTAATCGCACCATTACCTATAC                   GTTCTCAATCACGTCTATAGTCAGTCAGCATCCGCTTTTGAAAAAGCCGAGCCGGGCTAT       1501   ---------+---------+---------+---------+---------+---------+   1560           CAAGAGTTAGTGCAGATATCAGTCAGTCGTAGGCGAAAACTTTTTCGGCTCGGCCCGATA                   TATTTCCGCAAAAATACGCAATCTGGGTGTGGCAATGATACGGCGAGCAACCACGAAATG       1561   ---------+---------+---------+---------+---------+---------+   1620           ATAAAGGCGTTTTTATGCGTTAGACCCACACCGTTACTATGCCGCTCGTTGGTGCTTTAC                   TTTGGTAAATACATTATTGACTCTGTCACTTACTGGGCGAAAAATTATGATATTGACGGC       1621   ---------+---------+---------+---------+---------+---------+   1680           AAACCATTTATGTAATAACTGAGACAGTGAATGACCCGCTTTTTAATACTATAACTGCCG                   TTCCGTTTTGATGAAATGACACTTTTAGACAGCACGACTATGAATAAACTGCGTGCCGCA       1681   ---------+---------+---------+---------+---------+---------+   1740           AAGGCAAAACTACTTTACTGTGAAAATCTGTCGTGCTGATACTTATTTGACGCACGGCGT                   CTGACAGCACTTGACCCACATATTATCATGTATGGCGAAGGTTGGGGCGATTCTAATGCA       1741   ---------+---------+---------+---------+---------+---------+   1800           GACTGTCGTGAACTGGGTGTATAATAGTACATACCGCTTCCAACCCCGCTAAGATTACGT                   AATAATATTCCTGAAACGTCTATCAATAATTACAAAAATGTTCCTGGTATTGGTTTTTTC       1801   ---------+---------+---------+---------+---------+---------+   1860           TTATTATAAGGACTTTGCAGATAGTTATTAATGTTTTTACAAGGACCATAACCAAAAAAG                   AATCCTGGTGAACGTGACGCCATCAGTAATAATGGAGGTTCTGCAGGTGGATTCGCAGCA       1861   ---------+---------+---------+---------+---------+---------+   1920           TTAGGACCACTTGCACTGCGGTAGTCATTATTACCTCCAAGACGTCCACGTAAGCGTCGT                   GGAAATACGGCAAGTACTATAACGGTCGCAGGAGCACTTTTGGCTTCTGGAGGTTGGAAC       1921   ---------+---------+---------+---------+---------+---------+   1980           CCTTTATGCCGTTCATGATATTGCCAGCGTCCTCGTGAAAACCGAAGACCTCCAACCTTG                   GGAAATGGCACAGTGCAAGCTTTTTTGACGCCAAGCCAGTCTATCAATTATGTTGAATGT       1981   ---------+---------+---------+---------+---------+---------+   2040           CCTTTACCGTGTCACGTTCGAAAAAACTGCGGTTCGGTCAGATAGTTAATACAACTTACA                   GATGATAGCTTCACCTTAAATGACTCACTCTGGTCAGCTGATCCGAATGATTCTGTGGCG       2041   ---------+---------+---------+---------+---------+---------+   2100           GTACTATCGAAGTGGAATTTACTGAGTGAGACCAGTCGACTAGGCTTACTAAGACACCGC                   ACACATCAAGCGCGAGTGACGCTCGCAAATGCCACGAATATTCTTGCAAACGGTGTGACA       2101   ---------+---------+---------+---------+---------+---------+   2160           TGTGTAGTTCGCGCTCACTGCGAGCGTTTACGGTGCTTATAAGAACGTTTGCCACACTGT                   TTTATGGAAACAGGACAGGAGTTTGACCAGTCCAAATTAGTCAATCCGTCAAATCTGACG       2161   ---------+---------+---------+---------+---------+---------+   2220           AAATACCTTTGTCCTGTCCTCAAACTGGTCAGGTTTAATCAGTTAGGCAGTTTAGACTGC                   CCGCTCTCCCCTACACAGACACAGGCTTATCAATCGGGTAGTATGGAAAAACCTGCATGG       2221   ---------+---------+---------+---------+---------+---------+   2280           GGCGAGAGGGGATGTGTCTGTGTCCGAATAGTTAGCCCATCATACCTTTTTGGACGTACC                   TATCCTGCGTCATGGGATACTGCCAAAAATTCATACAATGGACTTTTTGGCTTGGCTAGT       2281   ---------+---------+---------+---------+---------+---------+   2340           ATAGGACGCAGTACCCTATGACGGTTTTTAAGTATGTTACCTGAAAAACCGAACCGATCA                   AATGGTACTTACTATGGCAATTATTGGCCAGGAAGTAATCTCTACACCCCAGTAGTTGCG       2341   ---------+---------+---------+---------+---------+---------+   2400           TTACCATGAATGATACCGTTAATAACCGGTCCTTCATTAGAGATGTGGGGTCATCAACGC                   GGGGATGTAGTCAATGCCATGAATTGGGATAATGTCAAGGACAATCAAAATGCTGTTAAT       2401   ---------+---------+---------+---------+---------+---------+   2460           CCCCTACATCAGTTACGGTACTTAACCCTATTACAGTTCCTGTTAGTTTTACGACAATTA                   TTTATTGGAAATTTGATGAAATTTAAAAAATCCAATCCGCAATTTTGGCCTGATGATTAT       2461   ---------+---------+---------+---------+---------+---------+   2520           AAATAACCTTTAAACTACTTTAAATTTTTTAGGTTAGGCGTTAAAACCGGACTACTAATA                   AGCAAACTTGCTTGGACTCCTACCAGTATAGGTGTAGAAAATGTCACAAATGCGTCAAAC       2521   ---------+---------+---------+---------+---------+---------+   2580           TCGTTTGAACGAACCTGAGCATGGTCATATCCACATCTTTTACAGTGTTTACGCAGTTTG                   GGAGTGATCACAGAAGAATTAACATCGGGTGCGACCAAATATTTAGTCATATTGAATGCT       2581   ---------+---------+---------+---------+---------+---------+   2640           CCTCACTAGTGTCTTCTTAATTGTAGCCCACGCTGGTTTATAAATCAGTATAACTTACGA                   AGCGGTAATTCGGTCAAAATTGGTCAAGGTGGTCAGTTTTATGGGCTGTCAAATTTGACA       2641   ---------+---------+---------+---------+---------+---------+   2700           TCGCCATTAAGCCAGTTTTAACCAGTTCCACCAGTCAAAATACCCCACAGTTTAAACTGT                   GGTAAAACTGTGATAATTTCTGACGATAGTAGCTTAACAGCTAATCAAGTTTTGAATAGC       2701   ---------+---------+---------+---------+---------+---------+   2760           CCATTTTGACACTATTAAAGACTGCTATCATCGAATTGTCGATTAGTTCAAAACTTATCG                   TCTGTCACTCTGTCAAATTTGACAGTCACCGTTATCCAACTTTCTAAATAA       2761   ---------+---------+---------+---------+---------+-   2811           AGACAGTGAGACAGTTTAAACTGTCAGTGGCAATAGGTTGAAAGATTTATT          
 
         [0009]     Ways of producing the novel plasmidic gene encoding the novel amylolytic enzyme which is secreted into the environment, according to the invention, are as follows: the plasmid DNA, previously isolated from the bacterial strain, favorably belonging to  Lactococcus  genus, encoding the amylolytic enzyme, is digested with restriction enzymes, favorably EcoRI, SacII, SalI, SmaI, SpeI, XhoI or XbaI. Subsequently, the excised fragment, favorably of size not smaller than 3.0 kb is ligated with a plasmid capable of replicating in bacterial cells, favorably those of  Lactococcus  genus, especially pIL253, pGKV210 or pIL252 also digested previously with restriction enzymes, favorably with EcoRI, SalI, or SmaI, after which the two DNA fragments are recombined together and introduced by means of electroporation into bacterial cells, which are grown in a known manner, and from the cultivated population, cells possessing the novel gene encoding the novel amylolytic enzyme are isolated in a known way.  
         [0010]     In the method according to the invention bacterial strains into which the recombined DNA is introduced are favorably used  Lactococcus lactis  MG1363,  Lactococcus lactis  IL1403 or  Lactococcus lactis  IBB140.  
         [0011]     In the method according to the invention colonies producing the amylolytic enzyme and carrying the novel gene are positively separated by inoculation into fresh liquid medium with antibiotic and starch if necessary.  
         [0012]     The novel enzyme secreted into the environment, encoded by the novel plasmidic gene according to the invention, has the following amino acid structure (SEQ. ID NO. 5):  
                                 1   MKKTKLRICL FPIVLLISFT LLSNGTGKQI VFATSSSEYA TQALSDEKNA                    51   TQNNDFTSFD IKWAYEGTDL GFNYSTTSTT FKIWSPTATS VQLISYGTNT               101   NPTAAQVSAK AMTRGTSATP TNHATNTIGV WTLTVPGNQN GMVYAYKLTF               151   ADGTVSDYAG STYGTLSTSS VSNTTNDPYS IATTQGGNRS VVESSANLAS               201   NLALAQGKSA TWRVASPTQA IVDELHIRDF TSSSTSGVSA GNRGKFLGVI               251   QSGTTDPNTG TATGLDYLKN EGFNYIQLHA SQSVCVGQRS GNRTTAQPNN               301   FNWGYDPQMK WYQSEYASNS VNPVTRINEM KEMVQGLHTN GISVVMDMVL               351   NHVYSQSASA FEKAEPGYYF RKNTQSGCGN DTASNHEMFG KYIIDSVTYW               401   AKNYDIDGFR FDEMTLLDST TMNKLRAALT ALDPHIIMYG EGWGDSNANN               451   IPETSINNYK NVPGIGFFNP GERDAISNNG GSAGGFAAGN TASTITVAGA               501   LLASGGWNGN GTVQAFLTPS QSINYVECHD SFTLNDSLWS ADPNDSVATH               551   QARVTLANAT NILANGVTFM ETGQEFDQSK LVNPSNLTPL SPTQTQAYQS               601   GSMEKPAWYP ASWDTAKNSY NGLFGLASNG TYYGNYWPGS NLYTPVVAGD               651   VVNAMNWDNV KDNQNAVNFI GNLMKFKKSN PQFWPDDYSK LAWTPTSIGV               701   ENVTNASNGV ITEELTSGAT KYLVILNASG NSVKIGQGGQ FYGVSNLTGK               751   TVIISDDSSL TANQVLNSSV TLSNLTVTVI QLSK          
 
         [0013]     This enzyme exhibits a unique set of profitable features, especially showing activity in a wide range of acid pH from 3.5 to 5.5 as shown on graph ( FIG. 1 ) while its optimum is at a pH 4.4. At the same time the optimal temperature of the enzyme&#39;s activity is in a range from 350-45° C. as shown on graph ( FIG. 2 ), and is thermally inactivated at temperatures higher than 50° C. as shown on graph ( FIG. 3 ).  
         [0014]     Application of the amylolytic enzyme encoded by the novel gene according to the invention, in fermentation of plant material, favorably starch, pullulan, amylose, amylopectin, feed production, obtaining glucose, lactic acid and production of probiotic specimens, probiotic-containing food, feed and fodder, according to the invention is based on the fact that its gene is introduced into the structure of the bacterial strain, favorably the strains of  Lactococcus lactis  IBB500, IBB501, IBB502, IBB140 and subsequently of applying the strains obtained in fermentations of plant material or milk or in biomass production.  
         [0015]     The gene according to the invention has been sequenced and the sequence obtained indicates that it belongs to the family of pullulanase encoding genes. The analysis of the gene&#39;s promoter region as well as functional studies suggest that the expression of this gene undergoes catabolic repression.  
         [heading-0016]     General Characteristics of the Amylolytic Enzyme  
         [0017]     From the comparison of features of pullulanases from studied  L. lactis  IBB500 and IBB502 strains it can be concluded that we are dealing with an enzyme produced in two organisms. Pullulanase deriving from both of the strains had the optimum pH of 4.4 and reached the optimal temperature at 45° C. ( FIGS. 1, 2 ).  
         [0018]     One should note the rather narrow pH range of the enzyme&#39;s activity between pH 3.5-5.5 as well as the fact that it is significantly lower than for pullulanases deriving from other bacteria (Ara K., Igarashi K., Saeki K., Kawai S., and S., Ito. 1992. Biosci. Biotech. Biochem. 56:62; Kim Ch., Nashiru O., J., Ko. 1996. FEMS Microbiology Letters 138:147; Takasaki Y. 1987. Agric. Biol. Chem., 51:9).  
         [0019]     The enzyme obtained with the help of the novel gene according to the invention, shows homology to amylolytic enzymes of the pullulanase group and the highest homology was found to pullulanase from  Termotoga maritima.  In the amino acid sequence of pullulanase from  L. lactis  IBB500 four conserved motives, characteristic for many amylolytic enzymes were identified. The nucleotide sequence of pul gene was preceded by a RBS typical for  Lactococcus  in front of which a long 500-nucleotide non-coding region was identified.  
         [0020]     The thorough analysis of the non-coding region upstream of the pul gene showed the presence of several putative promoter sequences. Due to the earlier observations of the effect of glucose on pullulanase production, from which was concluded that the expression of the pul gene may be regulated by catabolic repression, the promoter region was analyzed for the presence of the cre motive,. characteristic for this mechanism of regulation (Weickert M. J. and S. Adhya. 1992. J. Biol. Chem. 267: 15869). It was determined that the sequence homologous to the 14-nucleotide cre sequence is present in the analyzed promoter region. This observation strongly supported the earlier hypothesis. Another argument for the validity of this hypothesis was acquired during experiments on the influence of glucose as well as starch and its derivates on the pullulanase production in the IBB500 strain. In the process it was determined that starch and its derivatives induce pullulanase production although at different levels, while in the presence of glucose the repression of production of this enzyme is observed (Domań M., E. Czerniec, Z. Targoński and J. Bardowski. 2000. Food Biotechnology 17:67, Elsevier Science B.V., Amsterdam).  
       HERE BELOW THE EXAMPLES OF REALIZATION ARE PRESENTED  
       [heading-0021]     Materials and Methods  
         [heading-0022]     Bacterial Strains, Growth Conditions and Plasmids Used.  
         [0023]     Bacterial strains and plasmids used in these studies are shown in Table 1.  Lactococcus lactis  strains were grown in BHI medium (Oxoid, England) or M17 (Difco, USA), at the temperature of 28° C.,  Escherichia coli  in Luria-Bertani medium (LB), at the temperature of 37° C. Where necessary for selection, the following antibiotics were used: erythromycin—5 μg/ml for  L. lactis  and ampicyline—100 μg/ml for  E. coli.    
       Example I  
       [0024]     Plasmid DNA was isolated from  Lactococcus lactis  IBB500 bacterial strain encoding the amylolytic enzyme. Subsequently, it was digested with EcoRI restriction enzyme, then the excised fragment not smaller that 8.0 kb was ligated with a plasmid capable of replicating in  Lactococcus  bacterial cells—pIL253, digested previously also with EcoRI restriction enzyme. Both DNAs were recombined with each other and introduced by the method of electroporation into  Lactococcus lactis  cells which were grown and from the cultivated population cells carrying the novel gene of the structure according to the invention were isolated by inoculation into fresh liquid medium with antibiotic and starch.  
         [0025]      L. lactis  cells were transformed by method of electroporation (Holo H. and I. F. Nes. 1989. Appl. Environ. Microbiol. 55: 3119).  
         [0026]     The remaining molecular biology techniques used in the example were carried out according to the standard methodology (Sambrook J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2 nd  edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).  
       Example II  
       [0027]     Plasmid DNA was isolated from  Lactococcus lactis  IBB500 bacterial strain encoding the amylolytic enzyme. Subsequently, it was digested with XhoI restriction enzyme, the excised fragment of size not smaller than 5.0 kb was ligated with a plasmid capable of replicating in  Lactococcus  bacterial cells—pIL253, digested previously also with XhoI. Both DNAs were then recombined with each other and introduced by method of electroporation into  Lactococcus lactis  cells which were grown and from the cultivated population cells carrying the novel gene of the structure according to the invention was selected by inoculation into fresh liquid medium with antibiotic and starch.  
         [0028]      L. lactis  cells were transformed by method of electroporation (Holo H. and I. F. Nes. 1989. Appl. Environ. Microbiol. 55: 3119).  
         [0029]     The remaining molecular biology techniques used in the example were carried out according to the standard methodology (Sambrook J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2 nd  edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).  
       Example III  
       [0030]     The structure of the nucleotide sequence of the novel gene and the amino acid sequence of the amylolytic enzyme encoded by this gene according to the invention was determined by sequencing a Pstl 1.5 kb fragment of the pIBB502 plasmid, carrying a part of the novel gene, which was previously cloned in pBluescript plasmid (Stratagene), what generated the pIBB504 plasmid. This plasmid was used to sequence the cloned fragment from both sides. Transformation of  E. coli  cells was carried out according to the standard method with the use of CaCl 2  (Sambrook J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2 nd  edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).  
         [0031]     The complete nucleotide sequence of the fragment and of the lacking part of the novel gene encoding the amylolytic enzyme were obtained by using the sequencing method named “primer walking”. For DNA sequencing BigDye Terminator set (Promega, USA), PCR machine model 2400 (Perkin-Elmer) and sequencer ABI377 (Applied Biosystem, USA) were used. The nucleotide and amino acid sequences obtained were analyzed using the GCG programs (Genetics Computer Group. 1991. Program manual for the GCG package, version 7, April 1991. Genetic Computer Group, Madison, Wis., USA) and BLAST (Altschul S. F., W. Gish, W. Miller, E. W. Myers and D. J. Lipman. (1990), J. Mol. Biol. 215: 403).  
       Example IV  
       [0032]     For determining the amylolytic activity two tests—quantitative and qualitative were applied.  
         [heading-0033]     Qualitative Test on Plates  
         [0034]     Bacterial strains were plated on BHI with 0.5% starch and incubated in 28° C. for two days. Subsequently, the grown colonies were overlayed with Lugol reagent, which colors the non-degraded starch dark blue. A clear zone of non-colored medium surrounded colonies that secrete the amylolytic enzyme into the environment.  
         [heading-0035]     Quantitative Determination of Enzyme Activity  
         [0036]     In this test a modified method was used to determine the activity of extracellular amylase (Nicholson W. and P. Setlow. 1990. Sporulation, germination and outgrowth, p 433. W C. R. Harwood and S. M. Cutting (ed.), Molecular Biology for Bacillus. John Wiley and Sons Ltd., Chichester, United Kingdom).  
         [0037]     In this method the enzymatic activity was determined in the supernatant of the culture: to 200 μof this supernatant 800 μl of the substrate (0.025% starch in 10 mM Tris-HCl-3 mM potassium acetate-25 mM CaCl 2 , pH 4.4) was added and incubated for 30 min. at 37° C. The reaction was stopped by adding 400 μof Lugol reagent and the absorption was measured at wavelength of 620 nm [A 620 ]. As a control non-inoculated medium was used.  
         [0038]     One unit of amylolytic activity (1 U) was established as the amount of enzyme, which in the conditions of the test, generated the decrease of the substrate&#39;s absorption A 620  by 0.1 compared to the control.  
                                                                                         TABLE 1                           Strains and plasmids.            Strains and plasmids   Genotype   Source                      Lactococcus lactis              IBB 500   amy +  wild type strain   IBB PAN collection           isolated from plant material       IBB 501   amy +  MG 1363 with   this work           pIBB501       IBB 502   amy +  derivative of MG   this work           1363 with pIBB502       IBB 140   amy +  IBB140 with   this work           pIBB502       IL 1403   amy − , plamid-free   A. Chopin, France       MG 1363   amy − , plamid-free   M. Gasson, England              Eschercia coli              TG1   supE thi Δ(lac-proAB) hsd   (Gibson, 1984)           (F′ +  traD proAB lacl q             Z M15)       IBB 504   amy −  derivative of TG1   this work           with pIBB504            Plasmids            pIBB500   30-kb, wild type plasmid   this work           with amy +  gene       pIBB501   20-kb fragment of pIBB500   this work           with amy +  gene cloned in           pIL253       pIBB502   13-kb derivative of   this work           pIBB501 with amy +  gene       pIBB504   1.5-kb Pstl fragment from   this work           pIBB502 in pSKII+       pBluescript SKII+   Amp R , vector for cloning   Stratagene, USA           and DNA sequencing       pIL253   Ery R , vector for gene   A. Chopin, France           cloning                  
 
         [0039]