Abstract:
A β-Glucanase enzyme capable of hydrolytically cleaving mixed glucans is presented. The β-Glucanase is sufficiently stable under alkaline conditions for use in industrial cleaning processes, especially in the brewing industry.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This application is filed under 35 U.S.C. 371 and based on PCT/EP98/04564, filed Jul. 21, 1998. 
     This invention relates to an enzyme capable of hydrolytically cleaving mixed glucans, which are linked alternately by 1,3- and 1,4-β-glucosidic bonds, into oligosaccharides and to the microorganism which forms this enzyme. 
     Enzymes such as these belong to the class of endo-1,3-1,4-β-D-glucan4-glucanohydrolases (EC 3.2.1.73; lichenases) or endo-1.3-β-D-glucosidases (EC 3.2.1.39; laminarinases). For the purposes of the present invention, an enzyme of this type is referred to herein as β-glucanase or beta-glucanase. 
     2. Discussion of Related Art 
     Polymeric mixed glucans of the type mentioned above are present in varying amounts in virtually all cereal products. Enzymes capable of cleaving them are required above all in the food, beverage and animal feed industries, the textile industry and in the processing of starch (R. Borriss “μ-Glucan-spaltende Enzyme”, in H. Ruttloff: “Industrielle Enzyme”, Chapter 11.5, Behr&#39;s Verlag, Hamburg (1994)). One of the most important applications of β-glucanases is in the beverage and brewing industries where enzymes such as these are used for degrading malt and barley β-glucan. The enzymes used for this purpose normally emanate from  Bacillus subtilis , as described for example in German patent DD 226 012 A1, or from  Bacillus amyloliquefaciens , although β-glucanases from other microorganisms, for example  Achromobacter lunatus, Athrobacter luteus, Aspergillus aculeatus, Aspergillus niger, Disporotrichum dimorphosporum, Humicola insolens, Penicillium emersonli, Penicillium funiculosum  or  Trichoderma reesei , are also known. A commercial product intended for use in the brewing industry is marketed, for example, under the name of Cereflo® (manufacturer: Novo Nordisk A/S). 
     Hitherto known β-glucanases have pH optima in the weakly acidic to neutral range, so that their use is confined to processes which are carried out at those pH values. The problem addressed by the present invention was to extend the field of application of β-glucanases and to develop a β-glucanase which would be sufficiently stable under alkaline conditions for use in industrial processes carried out under conditions. 
    
    
     DESCRIPTION OF THE DRAWINGS 
     FIG. 1 depicts in one-letter code the amino acid sequence of the β-glucanase according to the invention (SEQ ID-NO.:1) obtained from  Bacillus alkalophilus  DSM 9956. 
     FIG. 2 depicts glucanolytic activity as a function of pH for the β-glucanase obtained from  Bacillus alkalophilus  DSM 9956 in Example 1 below. 
     FIG. 3 depicts depicts glucanolytic activity as a function of temperature for the β-glucanase obtained from  Bacillus alkalophilus  DSM 9956 in Example 1 below. 
    
    
     DESCRIPTION OF THE INVENTION 
     The present invention relates to an enzyme obtainable from  Bacillus alkalophilus  DSM 9956 which has the glucanolytic activity mentioned at the beginning, to the microorganism Bacillus alkalophilus DSM 9956 which produces a β-glucanase and to the gene encoding the ,β-glucanase from  Bacillus alkalophilus  DSM 9956 which was identified and sequenced (SEQ ID-NO.:2) in the course of the work culminating in the present invention. If desired, this gene may be cloned in known manner in other bacteria and the β-glucanase may be expressed there. Accordingly, the present invention also relates to host organisms containing the said gene obtainable by essentially microbiological processes. The amino acid sequence—derived from the sequence of the β-glucanase gene from  Bacillus alkalophilus  DSM 9956—of the β-glucanase according to the invention obtainable from that microorganism (SEQ ID-NO.:1) is shown in the one-letter code in FIG.  1 . The β-glucanase from  Bacillus alkalophilus  DSM 9956, including the signal peptide, which is split off by a signal peptidase after transport through the cell wall of the microorganism and which, according to comparisons with data known from the literature [M.E. Louw, S. J. Reid, Watson, Appl. Microbiol. Biotech. 39 (1993), 507-513], presumably comprises 31 amino acids, consists of 308 amino acids. The corresponding microorganism is gram-positive, its cell form is rodlet-like (width ca. 0.7 μm to 0.9 μm, length ca. 2.5 μm to 4.0 μm); it was deposited by applicants on 13.04.1995 in the DSM—Deutsche Sammiung von Mikrooganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, and has been given the number DSM 9956. 
     A β-glucanase according to the invention preferably has a homology of more than 70%, more particularly 75% to 99%, to the β-glucanase from  Bacillus alkalophilus  DSM 9956. The same applies to the basic gene. 
     The enzyme according to the invention is preferably used in the food industry, more especially in the beverage and brewing industry, more particularly for removing glucan and/or lichenan in the cleaning of membranes and other equipment in those industries. 
     The present invention also relates to a process for removing glucan and/or lichenan in the cleaning of membranes and other equipment in the food industry, more particularly the brewing industry, using a β-glucanase according to the invention. 
     EXAMPLES 
     Example 1 
     Chromosomal DNA from  Bacillus alkalophilus  C/M2-3 was partly digested with Sau3A and a fraction of 4 to 8 kb large fragments was isolated by gel electrophoresis. After ligation into the BamH1-site of the plasmid pMK4, an  E. coli —Bacillus shuttle vector [M. A. Sullivan et al., Gene 29 (1984), 21-26], it was transformed into competent  E. coli  DH5α cells. Recombinant clones with β-glucanase activity were identified by coloring with Congo Red on LB plates containing 0.2% lichenin (pH 8.5). 
     The β-glucanase was purified from the cell supernatant of a clone in  E. coli  DH5α pmK4. After dialysis of the cell-free supernatant against 20 mM sodium phosphate buffer (pH 7.5), the dialyzate was fixed to Q-Sepharose (Pharmacia) and eluted with a linear gradient of 0-1 M NaCl in 25 mM sodium phosphate buffer (pH 7.5 or pH 9.0). 
     The detection and determination of the glucanolytic activity was based on modifications of the process described by M. Lever in Anal. Biochem. 47 (1972), 273-279 and Anal Biochem. 81 (1977), 21-27. A 0.5% by weight solution of β-glucan (Sigma No. G6513) in 50 mM glycine buffer (pH 9.0) was used for this purpose. 250 μl of this solution are added to 250 μl of a solution containing the agent to be tested for glucanolytic activity and incubated for 30 minutes at 40° C. 1.5 ml of a 1% by weight solution of p-hydroxybenzoic acid hydrazide (PAHBAH) in 0.5 M NaOH, which contains 1 mM bismuth nitrate and 1 mM potassium sodium tartrate, are then added, after which the solution is heated for 10 minutes to 70° C. After cooling (2 minutes/0° C.), the absorption at 410 nm is determined against a blank value at room temperature (for example with a Uvikon® 930 photometer) using a glucose calibration curve. The blank value is a solution which is prepared in the same way as the measuring solution except that the glucan solution is added after the PAHBAH solution. 1 U corresponds to the quantity of enzyme which produces 1 μmole of glucose per minute under these conditions. 
     The specific activity of the enzyme thus obtained amounted to 4390 mU per mg protein whereas the activity of the starting solution was lower by a factor of 152. The enzyme was colored (silver coloring) as a homogeneous band in SDS polyacrylamide gel electrophoresis. 
     With the aid of marker proteins (cytochrome c, equine myoglobin, chymotrypsinogen, ovalalbumin, bovine serum albumin) as an internal standard, the molecular weight of the β-glucanase from  Bacillus alkalophilus  DSM 9956 was estimated by SDS polyacrylamide gel electrophoresis to be about 30,000. 
     In isoelectronic focusing (pH 3 to 9), the isoelectric point of the β-glucanase was found by activity coloring to be at pH 5.2. 
     EXAMPLE 2 
     pH Profile 
     The determination of glucanolytic activity at various pH values was carried but in a Davies universal buffer (21.01 g citric acid . H 2 O, 13.61 g KH 2 PO 4 , 19.07 g Na 2 B 4 O 7 . 10 H 2 O, 12.11 g tris and 7.46 g KCl in 1 l dist. water; 50 ml of this stock solution are adjusted to the required pH with 0.4 N NaOH and made up with dist. water to 200 ml) at 40° C. after incubation for 30 minutes. As can clearly be seen from the pH profile shown in FIG. 2 (relative glucanolytic activity, rel. A., plotted against the pH), the enzyme is at its most active between pH 6 and pH 10.5. The optimum lies at pH 9. 
     EXAMPLE 3 
     Temperature Profile 
     The dependence on temperature of the glucanolytic activity of the β-glucanase obtained from  Bacillus alkalophilus  DSM 9956 was measured in glycine/NaOH at pH 9 after incubation for 15 minutes. The pH value of the test solution was adapted because the buffer has a dependence on temperature of ca. pH 0.033 per °C. The maximum of the glucanolytic activity is at 60° C., as shown in FIG. 3 where the relative glucanolytic activity (rel. A.) of the enzyme is plotted against the temperature (T). 
     Statement Under 37 C.F.R. §§ 1.821(f) and (g) 
     The contents of the attached paper Sequence Listing and its computer-readable form are the same and add no new matter in this application. 
     
       
         
           
             2 
           
           
             1 
             308 
             PRT 
             Bacillus alkalophilus DSM 9956 
           
            1
Met Lys Arg Lys Thr Phe Val Leu Phe Ser Leu Phe Thr Leu Leu Ile
1               5                   10                  15
Gly Met Phe Ser Thr Gly Phe Ala Asn Thr Gly Val Val Gln Ala Glu
            20                  25                  30
Asp Gly Arg Pro Met Gly Ser Thr Phe His Glu Thr Phe Asp Thr Phe
        35                  40                  45
Asn Thr Asp Arg Trp Ser Thr Ala Gly Val Trp Thr Asn Gly Ala Met
    50                  55                  60
Phe Asn Ala Thr Trp Tyr Pro Glu Gln Val Thr Ile Ser Asp Gly Lys
65                  70                  75                  80
Met Lys Leu Gln Ile Asp Lys Glu Asp Asp Glu Asp Ala Thr Pro Glu
                85                  90                  95
Tyr Lys Ala Gly Glu Leu Arg Thr Asn Gln Phe Tyr Gln Tyr Gly Leu
            100                 105                 110
Phe Glu Val Asn Met Lys Pro Ala Lys Ser Thr Gly Thr Val Ser Ser
        115                 120                 125
Leu Phe Thr Tyr Thr Gly Pro Trp Asp Trp Asp Asn Asp Pro Trp Asp
    130                 135                 140
Glu Ile Asp Ile Glu Phe Leu Gly Lys Asp Thr Thr Arg Val Gln Phe
145                 150                 155                 160
Asn Tyr Phe Thr Asn Gly Val Gly Asn Asn Glu His Tyr His Glu Leu
                165                 170                 175
Gly Phe Asp Ala Ser Glu Ser Phe Asn Thr Tyr Ala Phe Glu Trp Arg
            180                 185                 190
Pro Glu Ser Ile Ser Trp Tyr Val Asn Gly Glu Leu Val Tyr Thr Ala
        195                 200                 205
Thr Glu Asn Ile Pro Gln Thr Pro Gln Lys Ile Met Met Asn Leu Trp
    210                 215                 220
Pro Gly Ile Gly Val Asp Gly Trp Thr Gly Val Phe Asp Gly Glu Asp
225                 230                 235                 240
Thr Pro Val Val Thr Glu Tyr Asp Trp Val Arg Tyr Thr Pro Leu Glu
                245                 250                 255
Glu Leu Asp Asn Asn Gly Glu Gln Pro Lys Pro Val Val Pro Gly Lys
            260                 265                 270
Pro Glu Lys Pro Gly Lys Pro Gly Lys Asn Gln Lys Asn Gln Glu Asn
        275                 280                 285
Gln Glu Asn Gln Lys Asn Gln Glu Asn Gln Lys Asn Gln Lys Ile Arg
    290                 295                 300
Lys Thr Ser Ser
305
 
           
             2 
             927 
             DNA 
             Bacillus alkalophilus DSM 9956 
           
            2
atgaaaagga agacatttgt attattttct ttatttacgt tgttaattgg tatgttctca     60
acagggtttg caaatacagg tgtggttcag gcagaagatg ggagaccaat ggggtcgacg    120
tttcatgaaa cgtttgatac ctttaatacg gaccgctggt caacagctgg ggtatggaca    180
aatggagcaa tgtttaatgc gacatggtat ccagaacagg tgaccatttc agatgggaaa    240
atgaagttgc aaattgacaa ggaagatgat gaagatgcaa ccccagaata taaggctggg    300
gaattaagaa cgaatcagtt ttatcaatac gggttgtttg aagtcaatat gaagccagcg    360
aaatcaacag gaaccgtctc ttcactcttt acatatacgg gtccatggga ttgggataat    420
gatccttggg atgaaatcga tattgagttc cttggaaagg atacaacaag agtccaattt    480
aactatttta ctaacggagt aggaaacaat gaacattacc acgaattagg gttcgatgca    540
tcagaatctt ttaatacgta tgcttttgaa tggagaccag aatcaattag ttggtacgta    600
aacggagaat tagtatatac agcaacagaa aatatcccgc aaacaccaca aaaaattatg    660
atgaacttat ggcctggaat tggagtggat ggatggacag gcgtttttga cggagaagac    720
actccagttg taacggagta tgattgggta aggtacactc cactagagga attagataat    780
aacggagaac aaccgaaacc tgtagtgcca ggaaaaccag aaaaaccagg aaaaccaggg    840
aaaaaccaga aaaaccagga aaaccaggaa aaccagaaaa accaggaaaa ccagaaaaac    900
caaaaaatca gaaaaaccag tagttga                                        927