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 
     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, Lódź; 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). 
     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. 
     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). 
     It has been determined that besides strains from  Lactobacillus  genus, some strains of  Lactococcus  genus, naturally existing in the environment, also demonstrate this ability 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). 
     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 
     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 
     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 
               
               
                   
               
               
                   
                 GGGATTAGCGTGGTAATGGATATGGTTCTCAATCACGTCTATAGTCAGTCAGCATCCGCT 
               
               
                 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 
               
               
                   
               
               
                   
                 AATATTCTTGCAAACGGTGTGACATTTATGGAAACAGGACAGGAGTTTGACCAGTCCAAA 
               
               
                 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 
               
             
          
         
       
     
     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 
               
               
                   
               
               
                   
                 AAAATTTTTTTAAACAAAATGCACTAAAAGATTTTTTTGTGTTGCTGAGAGGTTGCAAAA 
               
               
                  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 
               
               
                   
                 CTAAACCCTAAATTGATAAGTTGTTGGTCATGATGAAAGTTTTAGACCAGTGGTTGACGC 
               
               
                   
               
               
                   
                 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 
               
             
          
         
       
     
     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. 
     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. 
     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. 
     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 
               
             
          
         
       
     
     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 35°-45° C. as shown on graph ( FIG. 2 ), and is thermally inactivated at temperatures higher than 50° C. as shown on graph ( FIG. 3 ). 
     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. 
     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. 
     General Characteristics of the Amylolytic Enzyme 
     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 ). 
     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). 
     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. 
     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 
     Materials and Methods 
     Bacterial Strains, Growth Conditions and Plasmids Used. 
     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 
     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. 
       L. lactis  cells were transformed by method of electroporation (Holo H. and I. F. Nes. 1989. Appl. Environ. Microbiol. 55: 3119). 
     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 
     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. 
       L. lactis  cells were transformed by method of electroporation (Holo H. and I. F. Nes. 1989. Appl. Environ. Microbiol. 55: 3119). 
     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 
     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.). 
     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 
     For determining the amylolytic activity two tests—quantitative and qualitative were applied. 
     Qualitative Test on Plates 
     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. 
     Quantitative Determination of Enzyme Activity 
     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). 
     In this method the enzymatic activity was determined in the supernatant of the culture: to 200 μl of 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 μl of Lugol reagent and the absorption was measured at wavelength of 620 nm [A 620 ]. As a control non-inoculated medium was used. 
     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