DEBRANCHING ENZYME PRODUCT, PREPARATION AND USE THEREOF

Abstract: A novel debranching enzyme product with properties in terms of thermostability and pH-optimum comparable to those of glucoamylase is produced by cultivating a strain belonging to the novel taxonomic group Bacillus acidopullulyticus. The novel debranching enzyme is used in conjunction with known saccharifying enzymes for the hydrolysis of starch.

Description: This invention is within the field of enzymes for the enzymatic conversion of starch to sugars. More spe cifically, the present invention relates to a novel debran ching enzyme capable of hydrolyzing alpha-1,6-glycosidic bonds in amylopectin and pullulan. This novel enzyme may be classified as a debranching enzyme of the pullulanase type. The invention is also directed towards a process for preparing the novel debranching enzyme and to the use thereof for the conversion of starch into dextrose and/or maltose containing starch hydrolysates, such as dextrose and maltose syrups. BACKGROUND OF THE INVENTION During the past decade the all-enzymatic hydrolysis of starch to syrups has gained wide and steadily increasing acceptance within the starch processing industry. Cn a world-wide basis the present enzymatic production of dextrose syrup from starch is estirnated to exceed 3 million tons (calculated as dry substance) per annum compared with about 0.4 million tons ten years ago. The process generally adopted for enzyme - enzyme hydrolysis-of starch encompasses the sequential steps of liquefaction and saccharification, the former being catalysed by an alpha-amylase, such as the thermostable B. licheniformis alpha-amylase, e.g. TERMAMYLR supplied by NOVO Industri A/S, Den- mark, and the.saccharJ.fication to dextxose (D-glucose) being ef ect- ed in the presence of a glucoamyLase, usually of f?zngal oricin,suc as AMG-150 L,also obtainable from the above named company. Obviously, the dextrose syrup producer aims at obtaining the highest possible yield of dextrose with the least possible expenditure of enzymes and of energy. The highest dextrose level attainable by the conventional process, starting with a 30-40 percent (by weight) starch suspension and saccharifying at 30 percent dry solids (D.S.), is about 96 percent (by weight) dextrose (96 DX). The reasons why the conventional starch cÏnversiÏn process does not proceed appreciably beyond that limit are essentially twofold. - Firstly, amylopectin (which constitutes about 802 of the majority of industrially important starches,including that of corn or maize), exhibits a branched chain structure in that it contains a significant further of alpha-l,6-glyc9sidic bonds. V7hereas amylopectin is only partially degraded by alpha'-amylase because alpha-amylase is practically devoid of alpha-1,6- glucosidase activity., substantial hydrolysis of the branched oligosaccharides including alpha-limit dextrins occurs in the subsequent saccharification step catalyzed by glucoamylase which also hydrolyses alpha-l,6-glycosidic links. However, the latter reaction proceeds at a considerably lower rate than the corresponding hydrolysis of alpha-l,4-bonds, whereby complete saccharification is impeded. Attempts to remedy the situation by adding more glucdamylase collides with a second obstructive feature (apart from incurring higher enzyme costs), namely the ability of glucoamylase also to catalyze dextrose polymerization (the so-called reversionary reaction). It should be mentioned in passing that an increase of starch conversion from about 96 DX:to about 98 DX (which for certain uses of dextrose is regarded as a highly significant improvemenX entailing thereby a reduction in the content of non-dextrose.contaminants by about'50 percent, - can be achieved by employing a relatively high level of glucoamylase combined with a dilution of the substrate to about 15 percent D.S., vide U.S. Patent No. 4,017,363. However, the subsequent concentration of such a dextrose solution to the higher conventional dry solids levels is energy consuing. The prior art has suggested employment of glucoamylase and a debranching enzyme simultaneously to obtain a significant increase in dextrose level, the rationale being that debranching enzymes have been shown to efficiently hydrolyse specific types of alpha-1,6-glycosidic bonds occurring in. branched chain oligosaccharides, and certain alpha-limit dextrins. In this respect reference is made to U.S. Patent No. 3,897,305 disclosing the combined use of glucoamylase and Aerobacter aeroqenes (Klebsiella pneumoniae) pullulanase, whereby a significant increase in DX of up to 2 percent can be achieved for syrups containing at least 30 percent D.S. Similar results have been demonstrated for the combined action of glucoamylase and another debranching enzyme, viz Pseudomonas amyloderamosa isoamylase as described in British Patent Application No. 8107287 However, in the first instance practically no saving of glucoamylase is achieved because the pH optimum of K. pneumoniae pullulanase makes it mandatory to conduct the saccharification at a relatively high pH (5,5-6) wherea ..tlle activity of glucoamylase is dramatically reduced. -- The same problem is not encountered with the isoamylase which has a pH optimum much closer to that of glucoamylase, whereby the dosage of the latter can be substantially reduced (by about 50 percent), simultaneously with the attain ment of an increase in DX value of 1-2 percent. However, a serious drawback of the isoamylase process (and actually shared by the known pullulanase process as well) is the heat lability of the debranching enzymes known in the art. This has meant that heretofore no saccharification in the presence of debranching enzyme has been technically feasible above about 55 C, whereas glucoamylase per se is adequately stable even at 600 C at which temperature level the risk of microbial contamination oz substrates is significantly reduced as com- pared with lower temperatures. Obstacles akin to those described hereinbefore have been encountered in the conversion of starch to high maltose syrup by means of beta-amylases. Like the alpha-amylases, beta-amylases are only capable of -partially degrading anWloPectin, in that hydrolysis thereof stops as an 1,5-alp'ZalJr = h point is approached. By combining the action of beta-amylase with that of a debranching enzyme, such as pullulanase or isoamylase, a substantial increase in maltose content can be achieved as disclosed in British Patent No. 1.144.956 and U.S. Patent No. 3,677,896. However, again saccharification temperatures above 550 C are not feasible due to the heat lability of the debranching enzymes', whereby the risk of bacterial contamination is substantially increased. It is an object of the present invention to obviate the shortcomings of the debranching enzymes known heretofore by furnishing a novel debranching enzyme having a temperature stability comparable to thatof glucoamylase, and .furthermore, possessing a pH optimum close to that' oflucoamlt.lase, The invention resides in the surprising discovery that a novel debranching enzyme of the pullulanase type having such properties is produced by newly discovered micro orgasms of the genus Bacillus belonging to the taxonomic group as hereinafter defined. SUMEIRIIY OF TIIE INVENTION According to its first aspect the presen'e inven- tion provides a debranching enzyme product which comprises. a novel debranching enzyme of the pullulanase type having the following characteristics: a) it is obtainable from a fermentation broth 'produced by cultivating in a suitable nutri ent medium a strain of the taxonomic group herein named Bacillus acidopullulyticus, b) it exhibits enzyme chemical properties essentially identical to and immunological properties identical or partially identical to those of the debranching enzyme derived from the Bacillus strain NCIB 11607, c) its activity optimum, measured by incubation for 30 minutes in acetate buffer (0.05 M) at pH 4 - 5 is at least about 600C, d) its pH optimum is in the range of 3.5 to 5.5 as determined by incubation for 30 minutes in acetate buffer (0.05 M) at about 600C, and e) it has a residual activity after 72 hours at 600 C as measured in a dextrose solution (30 percent D.S. by weight) at pH 5 of at least 50 percent. The debranching enzyme product may be in solid or liquid form and will generally have an activity in the range of from 10 to 350,000 pullulanase units ('as hereinafter defined) per g. In a preferred embodiment of the present invention the activity of the debranching enzyme product is in the range of from 100- to 15,000 pullulanase units per g. According to a further aspect of the present invention there is provided a process for the preparation of a debranching enzyme product comprising a debranching enzyme, which enzyme exhibits an activity optimum at about 600 C or above, a pH optimum in the pH-range of 3.5 to 5.5, and good thermostability at 600 C, which process comprises the cultivation in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts, of a debranch-- ing enzyme producing Bacillus strain belonging to the taxonomic group.herein named as Bacillus acidopullulyticus, or a debranching enzyme producing variant or mutant thereof, followed by recovery of said debranching enzyme product by conventional means. In a preferred embodiment of preparing the debranching enzyme product of this invention the Bacillus acidopullulyticus strain is selected from the group consisting of NCIB 11607, NCIB 11610, NCIB 11638, andCIB 11647. Most preferred strains are NCIB 11638 and NCIB 11647. In another preferred embodiment the Bacillus acidopullulyticus strain is NCIB 11611. In another preferred embodiment the Bacillus acidopullulyticus strain is NCIB 11636. In another preferred embodiment the Bacillus acidopullulyticus strain is NCIB 11637. In another preferred embodiment the Bacillus acidonullulticus strain is NCIB 11639. According to still another aspect the present invention provides a process for converting starch into syrups containing dextrose and/or maltose, which process comprises conducting the saccharification, optionally hut preLerably preceded hy a li.ciuefaction step to form a starch bydrolysate, in tic presence of an enzyme system which comprises effective amounts of the novel debranching enzyme as defined hereinbefore and a saccharifying enzyme selected from the group consisting of glucoamylase and beta-amylase. In a preferred mode of using the debranching enzyme of this invention the dry solids content of the starch hydrolysate is at least 30 percent by weight, the saccharification t'-hereof being conducted in the pH-range of from 3.5 to 5.5 at a temperature in the range of from 550 C to 65 C and preferably not more than 630C.' Preferred dosages of glucoamylase and beta-amylase are in the range of from 0.05 to 0.5 AG units and from 0.005 to 0.3 beta-amylase units, re snectively, the preferred dosage of¯debranching enzyme being in the range offran 0.005, to 5 pullulanase units (as hereinafter defined), per g of dry solids in the starch hydrolysate. fn an additional preferred mode the saccbarifying. enzyme is glucoamylase, whereby starch is converted into high DX dextrose syrup. DETAILED DESCRIPTION OF THE INVENTION The Microorganisms Isolation: The microoryanisms productive of the debranching enzyme of the present invention were selected by means of their ability to grow on a basal medium prepared by aseptic admixture of equal volumes of tryptone solution (1 percent) and a salt solution of the following composition: Salt Percent (NH4)2 SO4 0.04 MgSO41 2 0.1 CaCl2, 2H20 0.05 KH2P04 0.6, the pH of the salt solution being adjusted to 3 with sulphuric acid (lots) prior to heat sterilization. The pfl of the final basal medium was 4.8 - 5.2. Agar substrates were prepared from the basal medium containing pullulan or amylopectin (0.5 percent), with or without yeast extract (1 percent). Incubation was conducted at 30 C - 37 C. Pullulanase activity was detected as clearing zones following precipitation of pullulan by covering the agar plates with acetone. Debranching of amylopectin was detected as zones of hydrolysis exhibiting a strong blue colour with iodine-potassium iodide reagent. A number of microbial isolates from natural sources, particularly from soil samples, were selected according to the above screening procedure and, when subjected to further tests as hereinafter described, shown to produce the enzyme of the present invention. Examples of such cultures and mutants thereof were deposited with the National Collection of Industrial Bac.teria, Torry Research Station, Aberdeen, Scotland and accorded the reference numbers indicated in the following table I. Table I NCIB NO. Date of Deposition Origin 11607 8th Sept., 1980 Soil from Zoo, Penang, Malaysia 11610 8th Sept., 1980 Soil collected in Rio de Janeiro 11611 8th Sept., 1980 ibidem 11636 17th Febr.,.1981 Soil from citrus plan tation, Jamaica 11637 17 fah Febr., 1981 ibidem 11638 17th Febr., 1981 Mutant of NCIB 11607 11639 17th Febr., 1981 Soil collected in Hillerod, Denmark 11647 7th April, 1981 Mutant of NCIB 11607 Taxonomy The newly discovered microorganisms of the invention are aerobic, rod shaped and endospore forming bacteria. They therefore belong to the genus Bacillus. Their properties do not fit any of the recognized species of the genus Bacillus as described in Bergey's Manual (VIIIth Edition, WilliamsandWilkins, Baltimore, 1974), or the monograph: The Genus Bacillus by Gordon, Heynes and Pang, Agriculture Handbook No. 427, US Department of Agriculture, 1973. The present inventors therefore classify them as a hew taxonome group to which the name Bacillus acidopullulyticus has bean accorded. The diagnosis of this novel taxonolnic group is as follows: Morphology: Vegetative cells: Rods with a diameter of 0.6 - 1 micron. Swollen cells of protoplast cha racter are racter are frequently observed and seem to be stable during se veral hours of submerged fermentation. Spores. Cylindrical to ellipsoid, central to subterminal, scorangia not swollen. In phase contrast micro scopy the spores are difficult to distinguish from unstainable glo bules which may be present in the protoplasm. Contrary to these globules the spores. are stained by malachite green. Biochemical reactions: Gram reaction positive Catalase positive Aerobic growth positive Anaerobic growth negative Growth at 500 C negative Growth at 300 C-370 C good Growth in 3.5% NaCl negative Growth at pH t.8 - 5.2 in basal medium (vide sulrra! containing pul lulan as carbon source good Egg-yolk reaction negative Acid from: glucose positive mannitol positive Reduction of nitrate to nitrite positive Use of citrate negative Use of propionate negative Decomposition of tyrosine negative Production of a starch debranching pullulanase active at 60 C in the pH-range from 3.5 to 5.5: positive VP reaction variable Hydrolysis of casein variable Acid from: xylose variable arabinose variable The morphology of the new debranching enzyme producing strains indicates that they belong to the morphological group I of the genus Bacillus. A type culture of the new taxonomic group is NCIB 11607. Other representative strains of the group are NCIB 11610, 11611, 11636 and 11637, and 11639. A variety of Bacillus species are known producers of pullulan-hydrolysing enzymes (Progress in Industrial Microbiology, vol. 15 (19.79),and Morgan F. J., Adams Y.. R., and Priest F. G.: J. Appl. Bacteriol. 46 p. 291 (1979)). However, none of the know species of Bacillus produce a pullulanase retaining its activity at 60 C at pH below 5.0. Hence, according to an additional aspect of the present invention there is provided a biologically pure culture of a bacterium belonging to the novel taxonomic group of Bacillus acidopullulyticus as hereinbefore defined. Determination of Pullulanase Activity One pullulanase unit (PU) is defined as the amount of enzyme which under standard conditions (temperature 600 C and pH 5.0) hydrolyses pullulan at a rate corresponding to the formation of reducing groups eouivalent to 1 Z mole of glucose per minute. A 4 percent by weight solution (1 ml) of pullulan (supplied by. Sigma Chemical Co.) in acetate buffer (0.1 M, pH 5) is preheated for 10 minutes at 600 C followed by addition of a solution (1 ml) of the enzyme dissolved in deionised water at a concentration correspor)dincj to 0.0 4 - 0.15 PU per ml. The reaction is stopped after 30 rnjJtes by a-3dition of carbonate buffer, pH 10 (3 ml of 0.5 M). The concentration of reducing groups liberated is then determined by means of the Somogyi-Nelson method (J.Bicl. Chem. 153 (1944) 375-80; Ibid. 160 (1945) 61-68). Preparation of debranching enzyme product A Bacillus strain capable of producing the debranching enzyme of the present invention is usually propagated on a solid substrate prior to its cultivation under aerobic conditions in a suitable fermentation medium. Both media contain assimilable sources of carbon (e.g. glucose for liquid and amylopectin for solid media), a basal salt composition (vide supra) comprising e.g. ammonium s-Lphate as the nitrogen source, together with growth promoting nutrients, such as yeast extract and/or corn steep liquor (liquid medium) or tryptone (solid substrate). The fermentation is typically conducted at slightly elevated temperature, generally in the range of from 300 C to 35- C and at a pH below 6, preferably in the range of 5.0 - 6.0 and preferably kept approxiinately constant by automatic means. The enzyme is excreted into the medium. The ensuing fermentation broth, usually containing from about 0.1 to about 50 PU per ml, may be freed of bacterial cells, debris therefrom together with other solids, for example by centrifugation. The supernatant containing the enzyme may be further clarified, for example by filtration or centrifugation, and then concentrated as required, for example by ultrafiltration or in an evaporator under reduced pressure to give a concentrate which, if desired, may be taken to dryness, for example'by lyophilization or spray-drying. Typically, the resulting crude enzyme product exhibits an activity in the range of from 100 to 15,000 PU per gram. Purification of Debranching Enzvme The debranching enzyme of the present invention may be purified from a crude enzyme product, such as the concentrate obtained in Example 2 hereinafter,for example by a combination of cation and anion exchange chromatography. A column of CM-Sepharose CL-6B was equilibrated with a phosphate-citrate buffer (0.05 M Na2HPO4 titrated with 0.05 M citric acid to pH 4.5). The enzyme concentrate was diluted with water to the same conductivity as the equilibrating buffer and the pH was adjusted to 4.5. The sample was then applied to the column, whereby the enzyme was completely adsorbed. Elution was conducted with the same buffer linearly admixed in a gradient mixer with 0.05 M solution of Na2HPO4 so as to provide a gradient in pH. The fractions were analysed for pullulanase activity by the method described hereinbefore and for protein content by the Lowry method (J. Biol. Chem. 193 (1951) 256), and fractions showing enzyme activity were pooled. A column of DEAE-Sepharose CL-6B was equilibrated with phosphate-citrate buffer (0.01 M Na2HPO4 titrated with 0.01 M citric acid to pH 6.5). The pool was diluted to the same conductivity as the equilibrating buffer, adjusted to pH 6.5, and then applied to the column. The completely adsorbed enzyme was eluted with the same buffer admixed with phosphate-citrate buffer (0.15 M Na2HPO4 titrated to pH 6.5 with 0.15 M citric acid) thereby yielding a linear gradient in buffer concentration. Fractions were collected and analysed as before. The peak fractions showed a specific activity of about 350.000 PU/g. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (S.G. Braun et al., J. Virology vol. 10 (1972) 221) the contents of the fraction with highest activity exhibited in single protein band corresponding to MW about 100,000 Dalton. The protein has a pI of 5.0 as determined by isoelectric focusing on "LKB Ampholine PAG" plates (following the instructions given by LKB-Produkter AB, Sweden). Enzyme Chemical Properties The dependence of the activity of the debranching enzyme of this invention on pH in the range of 3.5 - 5.5 at different temperature levels was determined by the method for determination of pullulanase activity described above, using a reaction mixture of which pH and temperature were adjusted to predetermined values. Reference is now made to the attached drawing graphically illustrating the relative activity plotted against pH at 550C, 600C and 650c, respectively. To determine the thermostability of the enzyme in glucose syrup (ca. 30 percent D.S.) a solution of the enzyme product prepared in the following Example 1 was dissolved in deionised water to give a solution containing 3 PU per ml. Aliquots (1 ml) of this solution were mixed with 1 ml of 0.1 M citrate-phosphate buffer and glucose (0.8 g). After incubation of samples for 3 days at 50 0C and 600C the residual activity was determined by means of the assay for amylopectin debranching activity. A comparative test was conducted with Pseudomonas isoamylase (Hayashibara Biochemical Laboratories, Japan, containing 500,000 isoamylase units (IA) per g) diluted to 300 IA per ml. ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ The results are presented in the following table II, Table II EMI17.1 <tb> <SEP> Percent <SEP> residual <SEP> activity <SEP> afterj <tb> <SEP> 3 <SEP> days <tb> Temperature <SEP> pH <SEP> after <SEP> incu- <SEP> Debranching <SEP> enzyme <SEP> Isoamylase <tb> <SEP> C <SEP> bation <SEP> of <SEP> present <SEP> in- <tb> <SEP> vention <tb> <SEP> 50 <SEP> 4.5 <SEP> 109 <SEP> 75 <tb> <SEP> 4.9 <SEP> 100 <SEP> 68 <tb> <SEP> 4.9 <SEP> 89 <SEP> 1 <tb> <SEP> 60 <SEP> ¯ <SEP> <tb> <SEP> 5.2 <SEP> ... <SEP> 85 <SEP> 3 <tb> Assay of Amylopectin Debranching Activity: : Hydrolysis of the alpha-1,6-bonds of amylopectin causes an increase in the colour intensity (as measured at 610 nm) of the blue iodine-amylopectin complex. This increase is dependent on the amount of alpha-1,6-bonds hydrolysed. The enzyme solution (1 ml), diluted in deionized water to a concentration corresponding to 1-2 PU and 20 - 40 IA per ml for debranching en7.yme and isomylase, respectively, is mixed with acetate buffer (1 ml of 0.5 M, p11 4.5) and a 1 percent solution (5 ml) of amylopectin (CPC, SNOtffLAKE 04201 starch). The mixture is incubated for 30 minutes at 50 C. An aliquot (0.5 ml) is mixed with 15 ml of 0.02 N Il2SO4 and 0.5 ml of iodine solution (0.01 M iodine in 0.2 percent potassium iodide). After 15 minutes at room temperature the optical density at 610 nm is compared with that of a blank run with heat inactivated enzyme. Immunological properties Monospecific antiserum was generated by immunizing rabbits with purified debranching enzyme according to the method described by N.H. Axelsen et al., A Manual of Quantitative Immunoelectrophoresis (Oslo 1973) chap. 23. The debranching enzyme was prepared by cultivating the strain NCIB 11638 (cf. Example 1 hereinafter provided) and purified as described above. The immunogen (0.5 ml of a solution containing 18 PU per ml, corresponding to 0.15 mg of protein per ml) was mixed with Freund's adjuvant (0.5 ml) and injected subcutaneously into rabbits every second week. Antiserum was'obtained after a total immunization period of 8 weeks and immunogiobulin was prepared therefrom as described byN.H. Axelsen et al., supra. Two samples of the crude debranching enzyme product derived from the sazle strain (NC1B 11638), e.g. as obtained according to Example 1 hereinafter (each sample containing 10 PU per ml), were subjected to crossed immunoelectrophoresis according to the same authors (chap. 3) First dimension: 1 percent agarose in TRIS-boric acid buffer (20 mM, pH 8.6); electrophoresis at 150C, 8 volts per cm for 2 hours. Second dimension: Same agarose gel as above containing the immunoglobulin (100 ill); electrophoresis at 150C, 1 volt per cm for 16 hours. One plate, stained with Coomassie Brilliant Blue, gave a single peak of immunoprecipitate. The second plate was used for detecting the enzyme by means of an overlayer technique. The plate was covered with a gel containing pullulan and agarose (1 percent of each) in maleate buffer (0.1 M, pH 5). After incubation for 2 hours at 50 0C the remaining pullulan was precipitated with acetone whereby a clearing zone indicating pullulan hydrolysis became visible. Comparison of the two electropherograms showed that the clearing zone and the stained immunoprecipitate were superimposable, thus demonstrating the monospecificity of the immunoglobulin generated against the debranching enzyme produced by the strain NCIB 11638. The immunological properties of the debranching enzyme derived from each of the other deposited strains were compared with those of the enzyme from NCIB 11638 by crossed immunoelectrophoresis, using the above prepared immunoglobulin as reference antiserum. The immunograms so obtained demonstrated the identity or partial identity with respect to immunochemical properties of all specimens of debranching enzyme investigated. With respect to the meaning of the terms "immunochemical identity" and "partial immunochemical identity", reference is made to the above cited monograph by N.H. Axelsen et al., chapters 10 and 11, respectively. The following examples are presented as illustrative embodiments of this invention and are not intended as specific limitations thereof. Example 1. Preparation of Debranching Enzyme Product from Strain NCIB 11638 The strain NCIB 11638 (which is a mutant of NCIB 11607) was propagated on agar slants for 2 days at 370 C. The agar medium was prepared from the basal medium (p.9) by addi- tion of amylopectin(0.5percent of Snow Flake waxy starch CPC) and 2 percent Bacto-agar (difco). The cells were suspended in sterile water and transferred to a 2 liter laboratory fermentor (Eschweiler, -Kiel, - West Germany) containing 1 liter of the following medium: Yeast extract 0.2 percent Corn steep liquor (50% dry substance) 1.0 - - MgSO4.7H 0 0.025 K2HPO4 0.05 (NH4)2SO4 0.125 CaCl.2H2O 0.025 PLURONIC J. 61 0.1 dissolved in tap water The pH was adjusted to 5.5 prior to autoclaving at 130 C for 30 minutes. Glucose was autoclaved separately and added to a final concentration of 1 percent. During the cultivation pH was kept at 5.5 + 0.2 by addition of sulphuric acid (2 percent solution). The tempera- ture was 30.0 -+ 0.10 C, and the aeration rate 650 ml per minute (# 10 percent) After 22 hours of cultivationt substrate exchanye was initiated at a dilution rate of' 0.03 hour 1 After a further 36 hours steady state fermentation conditions were reached at a cell density of about 4 g of dry cells per litre (corresponding to an absorbance of 8 at 450 nm). The pullulanase activity was 7.5 PU per ml. During 6 days of steady state continous fermentation, 4,000 ml of culture liquid was collected in a chilled flask. The cells were removed by centrifugation for 30 minutes at 2,300 rpm, and the supernatant,containing 5.1 PU per ml,was concentrated by ultrafiltration through-a G-600 membrane using a standard laboratory outfit (supplied by Sartorius). The volume of this concentrate (800 ml) was further reduced in a Büchi rotating evaporator at 45 C. The final concentrate (76 ml) so obtained was frozen and lyophilized, thereby ytelc'.ing a dry powdered product (6.8 g) having an activity of 1150 PU per g. Example 2. Preparation of Debranching Enzyme Product from Strain NCIB 11647 . The fermentation was conducted in a stainless steel pilot fermentor (total capacity 550 litres, 70 cm diameter) provided with two 6-blade, 24 cm diameter, turbine agitators and with accessories required for continuous fermentation, including cquipment for the maintenance of constant pH and temperature. The fermentor was charged with a medium (300 litres) of the following composition: Soy bean meal 25 g Corn steep liquor (o% dry solids) 5 Potato starch 8.3 KH2P04 1.7 (NH4)2S04 1.0 Bacterial amylase BAN 120 L 0.013 ml Antifoam agent PLURONIC L 61 0.233 Tap water to 1 liter The pH of the medium was adjusted to 5.8 with sodium hydroxide solution. The starch was liquefied by raising the temperature gradually from 60 C to 90 C in the course of 50 minutes followed by sterilization at 121 0C for 60 minutes. A culture of the strain NCIB 11647, grown for 4 days in a Fernbach culture flask on the same nutrient agar substrate as used in Example 1, was used for inoculation of 300 litres of medium. The fermentation was conducted at 290C - 31 0C, the agitator speed being 100 r.p.m. and the aeration rate 150 standard liters per minute at a pressure of 0.5 bar. Starting at 33.5 hours after inoculation, a sterile medium of the same composition as above and prepared in the same manner was fed continuously to the culture at a rate of 15 liters per hour. When a volume of 375 liters was reached, automatic withdrawal of culture liquid was initiated. Approximately 30 hours after inoculation vigorous growth was observed by microscopy and as indicated by increased generation of CO2. Collection of culture broth commenced at a culture age of 73 hours. The debranching enzyme activity had then increased to above 10 PU per ml. During the period of from 115 hours to 210 hours an approximately steady state was maintained at.an enzyme concentration corresponding to between 50 and 76 PU per ml, the pH of the broth varying between 5.4 and 5.9. All subsequent operations were conducted at pH 5.5 - 5.7. The fermentation broth collected during 110 hours (1650 liters) was filtered on a rotary vacuum filter 2 (2.5 m ) coated with diatomaceous earth filter aid. The filtrate was concentrated and freed from low molecular 2 weight compounds by ultrafiltration using a DDS (7 m ss module (supplied by DDS, Copenhagen, Denmark) provided with a cellulose acetate membrane, type 600. The final concentrate (70 liters) contained 9.4 percent dry substance. Further concentration (to a volume of 21 liters), was effected in a rotary vacuum evaporator. An additional pressure filtration, using the same filter aid as above, gave an enzyme concentrate (15.5 kg, containing 28.8 percent dry substance) with an activity of 1500 PU per g. Example 3 Preparation of debranching enzyme product from strains NCIB 11610, NCIB 11636 and NCIB 11637. The strains were propagated and the fermentations conducted in the same manner as that described in Example 1. Maltose (0.5 percent) was used instead of glucose as the carbon source. Mean values of other parameters of the fermentations will appear from the following Table 1. ¯ Table 1 NCIB NCIB NCIB 11610 11636 11637 EMI24.1 <tb> <SEP> Dilution <SEP> rate, <SEP> 0.07 <SEP> 0.057 <SEP> 0.06 <tb> <SEP> hour <SEP> 1 <SEP> <tb> <SEP> Absorbance <SEP> at <tb> <SEP> 9.2 <SEP> 8.3 <SEP> 9.1 <tb> <SEP> 450 <SEP> nm <tb> baceria, <SEP> g/liter <tb> <SEP> bacteria, <SEP> g/liter <SEP> <tb> <SEP> Debranching <SEP> en <tb> <SEP> 0.80 <SEP> 0.25 <SEP> 0.11 <tb> <SEP> zyme <SEP> activity, <SEP> PU/ml <SEP> <tb> The culture broths were collected and the ebranching enzyme products were recovered essentially as described in Example 1. Details of the work-up procedurc will appear from the following Table 2. Table 2 EMI24.2 <tb> Strain <SEP> Culture <SEP> Supernatant <SEP> Lyophilized <SEP> product <tb> NCIB <SEP> No. <SEP> liquid <SEP> ml <SEP> ml- <SEP> PU/ml <SEP> g <SEP> PU/g <tb> 11610 <SEP> 5316 <SEP> 5110 <SEP> 0.37 <SEP> 7.5 <SEP> 69.2 <tb> 11636 <SEP> 1497 <SEP> 1405 <SEP> 0.08 <SEP> 4.4 <SEP> 14.4 <tb> 11637 <SEP> 4856 <SEP> 4790 <SEP> 0.04 <SEP> 6.0 <SEP> 12.2 <tb> Example 4 Preparation of Debranching Enzyme Product from strain NCIB 11639 Propagation of strain NCIB 11639, incubation and fermentation was conducted as described in Example 1. The fermentation medium had the following composition: *)Soy bean meal extract 2.0 percent Corn steep liquor 0.5 MgS04, 7H20 0.025 K2HP04 0.1 (NH4)2SO4 0.05 Amylase-liquefied starch (Dex trose equiv. 11) 0.5 PLURONIC L 61 0.1 dissolved in tap water. Extraction of soy bean meal solubies at pH 4.5 and 500C for 4 hours. Insoluble material was removed by centrifugation. The pH was adjusted to 4.0 before autoclaving at 130 C C for 60 minutes. During the cultivation pH was kept at 5.6 - 0.1 by addition of a sodium hydroxide solution (2 percent). The temperature was 30.0 - 0.1 C and the aeration rate 320 ml./min at a stirrer speed of 530 - 565 rpm. The fermentation was run continuously at a dilution rate of 0.-03 - 0.05 hr -1 for 186 hours when collection of over- flowing culture liquid was started. Culture liquid (5100 ml, 0.47 PU/ml) was collected ovcr'div ice for an additional 97 hours under the following conditions: Dilution rate 0.049 - 0.008 hr -l OD450 10.6 - 1.1 + Cell density 2.9 - 1.5 g/1 Debranching enzyme activity 0.5 # 0.1 PU/ml The cells were removed as described in Example 1. The supernatant was filtered on a Whatman GFA glass filter (11 cm) and then concentrated to 200 ml on an Amicon DC 2 hollow fibre module provided with an HlP10 membrane-. Turbidity was removed on the GFA filter and the filtrate further concentrated on the Amicon module using a 202-DDS 600 membrane (DDS, Copenhagen, Denmark) to yield of final concentrate (30 ml) with an activity of 65 PU/ml. The concentrate was stored deepfrozen. Example 5 100 kg of corn starch (Globe 03430, CPC) were ++ slurried with tap water containing 100 ppm Ca and the volume adjusted to 225 liters. The pH was adjusted to 6.3 and 135 g of TERMAMYL.R 60 L (NOVO Industri A/g, Denmark) were 60 added. This suspension was continously pumped through a jet cooker (Hydro-Thermal Corp. Milwaukee) where it was heated to 1050 C by direct steam injection and maintained at 1050 C for five minutes. The liquefied starch suspension was flash-cooled and pumped ove into a saccharification tank where it was held for about 1 hour at 950 C. The pH of the liquefied starch was adjusted to 4.0 at 950 C to stop'the reaction and the batch was then spray-dried without purification. The DE of the spray-dried maltodextrin was 6. Substrates for saccharification were prepared by redissolving suitable amounts of this maltodextrin in deionized water and making up to approximately 30 percent D.S. Aliquots of tis substrate were then taken and heated to, 600 C and the pH adjusted to 4.8. Different amounts of debranching enzyme were then added together with glucoamylase (Amyloglucosidase Novo 150, 150 AS units/ml). The reaction mixtures were sampled at set time intervals and the % dextrose in each sample determined by HPLC. The following results were obtained: EMI28.1 <tb> Debranching <SEP> Enzyme <SEP> Glucoamylase <SEP> Reaction <tb> PU <SEP> units/g <SEP> D.S. <SEP> AG <SEP> units/g.D.S. <SEP> time <SEP> (h) <SEP> pH <SEP> Dextrose <tb> <SEP> 0.225 <SEP> 72 <SEP> 4.1. <SEP> 95.8 <tb> <SEP> O <SEP> (control) <tb> <SEP> 0.225 <SEP> 96 <SEP> -4.1 <SEP> 95.9 <tb> <SEP> 0.113 <SEP> 72 <SEP> 4.6 <SEP> 93.3 <tb> <SEP> 0 <SEP> (control) <tb> <SEP> 0.113 <SEP> 96 <SEP> 4.3 <SEP> 94.4 <tb> <SEP> 0.01 <SEP> 0.150 <SEP> 96 <SEP> 4.3 <SEP> 96.1 <tb> <SEP> 0.1 <SEP> 0.113 <SEP> 96 <SEP> 4.4 <SEP> 95.9 <tb> <SEP> 0.5 <SEP> 0.113 <SEP> 96 <SEP> <SEP> 4.4 <SEP> 96; ;7 <SEP> <tb> <SEP> 1.0 <SEP> 0.113 <SEP> 72 <SEP> .4.6 <SEP> 97.0 <tb> <SEP> 2.0 <SEP> 0.113 <SEP> 72 <SEP> 4.7 <SEP> 97.3 <tb> <SEP> 4.0 <SEP> . <SEP> 72 <SEP> .113 <SEP> 72- <SEP> 4.7 <SEP> 97.4 <tb> - These. results indicate that when debranching enzyme is used together with glucoamylase, the same dextrose level can be obtained with half the glucoamylase dosage used in the control when a debranching.enzyme dosage 0.1 PU/g D.S. is used. - Xf the debranching enzyme dosage is increased the maximal obtainable dextrose level also increases. Example 6 Aliquots of the substrate prepared as in Example were were heated to 55 or 600 C and ,the pH adjusted to 4.9. An amount amount of ylucoamylase corresponding to 0.113 AG/g D.S. and an amount of debranching enzyme corresponding to 1.2 PU/g D.S. were added. The reaction mixtures were sampled and analyzed as in Example 5 Y The following results were obtained: EMI29.1 <tb> Temperature <SEP> Reaction <SEP> time <SEP> Dextrose <tb> <SEP> C <SEP> (hours) <SEP> % <SEP> <tb> <SEP> 24 <SEP> 84.5- <SEP> <tb> <SEP> 48 <SEP> 96.5 <tb> <SEP> - <SEP> 55 <SEP> <tb> <SEP> 72 <SEP> 97.1 <SEP> <tb> <SEP> 96 <SEP> 97.3 <tb> <SEP> 24 <SEP> 90.O <SEP> <tb> <SEP> 96.8 <SEP> <tb> <SEP> 60 <tb> <SEP> 72 <SEP> 97.0 <tb> <SEP> 96 <SEP> 97.3 <tb> These results confirm that the debranching enzyme-is sufficiently heat stable to be used at 60 C. Example 7 - A spray-dried maltodextrin substrate with a DE of 5 was prepared according to the method described in Example 5'. Aliquots of this substrate were heated to 60, 62.5 and 65 C and the pH adjusted to 4.9. An amount of glucoamylase corresponding to 0.113 AG/g D.S. and an amount of debranching enzyme corresponding to 1 PU/g D.S. were added. Control saccharifications towhich an amount of glucoamylase corresponding to 0.225 AG/g D.S. had bee added, but without debranching enzyme,were also included. The reaction mixtures were sampled and analysed as in Example b. The dry substance (D.S'.) during. sacchari- fication was 31 percent. EMI30.1 <tb> Debranching <SEP> <tb> <SEP> Enzyme <SEP> Glucoamylase <SEP> Temperature <SEP> Reaction <SEP> time <SEP> Dextrose <tb> <SEP> PU/gD.S. <SEP> ;.AG/g <SEP> D.S. <SEP> <SEP> C <SEP> (hours) <SEP> % <SEP> <tb> <SEP> 24 <SEP> 92.1 <tb> 0 <SEP> 0.225 <SEP> 60 <SEP> 48 <SEP> 95.3 <SEP> <tb> 72 <SEP> 96.0 <tb> 96 <SEP> 96.0 <SEP> <tb> 24 <SEP> 88.9 <tb> 1 <SEP> 0.113 <SEP> 60 <SEP> 48 <SEP> 96.1 <SEP> <tb> <SEP> 72 <SEP> 96.7 <tb> 96 <SEP> 97.0 <tb> 24 <SEP> 92.1 <tb> 48 <SEP> 95.1 <tb> <SEP> 0 <SEP> 0.225 <SEP> 62.5 <SEP> 72 <SEP> 95.8 <SEP> <tb> <SEP> 96 <SEP> 96.1 <tb> 24 <SEP> 89.3 <tb> <SEP> 48 <SEP> 95.9 <SEP> <tb> <SEP> 1 <SEP> 0.113 <SEP> 62.5 <SEP> 72 <SEP> 96.8 <tb> 96 <SEP> 97.0 <SEP> <tb> <RTI ID=30.10> 24 <SEP> 91.4 <tb> <SEP> 48 <SEP> 94.1 <tb> 0 <SEP> 0.225 <SEP> 65 <SEP> 72 <SEP> 95.2 <tb> 96 <SEP> 95.4 <SEP> <tb> 24 <SEP> 82.9 <SEP> <tb> <SEP> 48 <SEP> 89.9 <tb> 1 <SEP> <SEP> 0.113 <SEP> 65 <SEP> 72 <SEP> 92.8 <SEP> <tb> 96 <SEP> 93.6 <tb> These results indicate that saccharificaticn with debranching enzyme and glucoamylase may be carried out at 60-62.50 C. At 650 e the results are inferior, which may be due to glucoamylase instability at that temperature. Example 8 Aliquots. of the substrate prepared as in Example were heated to 60 C and the pH adjusted to 4.5, and 4.8. An amount of glucoamylase corresponding to 0.113 AG/g D.S. and an amount of debranching enzyme corresponding to 1 PU/g D.S. were added. The reaction mixtures were sampled and analy- zed as n Example 5 . The following results were obtained: EMI31.1 <tb> <SEP> Reaction <SEP> time <tb> <SEP> pH <SEP> (hours) <SEP> Dextrose <tb> <SEP> 24 <SEP> 89.6 <tb> 48 <SEP> 96.6 <tb> <SEP> 4.5 <SEP> 72 <SEP> 96.8 <tb> <SEP> 96 <SEP> 96.9 <tb> <SEP> 24 <SEP> 89.0 <tb> <SEP> d.8 <SEP> 48 <SEP> 96.5 <tb> <SEP> 4.8 <tb> <SEP> 72 <SEP> 96.8 <tb> <SEP> 96 <SEP> 97.0 <tb> These results indicate ;that similar results can be obtained when saccharifying at pH 4.5 and 4.8. Example 9 A 35 percent'D.S. (DE 6) substrate was prepared by dissolving a portion of the maltodextrin prepared as described in Example 5 in deionized water. Aliquots of this substrate were taken and heated to 600 C and the pH adjusted to 4.3 and 4.8. . An amount of glucoamylase corresponding to 0.113 AG/g D.S. and an amount of purified debranching enzyme (120 PU/mg protein) corresponding to 1 PU/g D.S. was added. The reaction mixtures were sampled and analyzed as in Example 5 . The following results were obtained: EMI32.1 <tb> Reaction <SEP> time <tb> <SEP> (hours) <SEP> - <SEP> pil <SEP> Dextrose <SEP> <tb> <SEP> .0 <SEP> 4.3 <tb> <SEP> 24 <SEP> ' <SEP> 4.3 <SEP> 89.2 <tb> <SEP> 48 <SEP> 4.2 <SEP> 96.1 <tb> <SEP> 72 <SEP> 4.2 <SEP> 96.2 <tb> <SEP> 24 <SEP> O <SEP> 4.8 <tb> <SEP> 24 <SEP> 4.5 <SEP> 88.4 <tb> <SEP> 48 <SEP> T.5 <SEP> 96.0 <SEP> <tb> <SEP> 72 <SEP> 4.4 <SEP> 96.2 <tb> . <SEP> . <SEP> <tb> These results indicate that similar dextrose levels can be obtained when saccharification is started at pH 4.3 or 4.8. Example 10 Substrates with different dry solids contents were prepared by dissolving 100 g of the DE 6 maltodextrin from Example 5 5 in different amounts of deionized water and adjusting the p's to 4.6 at 600 C. 0.113 AG/g D.S. of glucoamylase and 1 PU/g D.S. of debranching enzyme were added. The reaction mixtures were sampled and analyzed as in Example 5. The following results were obtained: EMI33.1 <tb> t <SEP> D.S. <SEP> % <SEP> Max. <SEP> Dextrose <tb> 25 <SEP> 9.7.7 <SEP> <tb> 30 <SEP> 97.2 <tb> 35 <SEP> 96;3 <tb> 40 <SEP> 94.9 <tb> 45 <SEP> 93.0 <tb> In a control saccharification without debranching enzyme, the maximum dextrose that could be obtained with tis substrate at 60 C, pH 4.5 and 0.225 AG units/g D.S. glucoamylase, and at 30 percent D.S., was 96.3. This means that saccharification may be carried out at a higher solids level when debranching enzyme is used to achieve the same dextrose level so that energy requirements for evaporation will be considerably less. Example ii A further batch of spray-dried maltodextrin with a DE of 5.0 was prepared according to the procedure outlined in Example 5. Substrates for saccharification were prepared by redissolving suitable amounts of this maltodextrin in'de- ionized water and making up to approximately 30 percent. Aliquots of this substrate were then heated to 600 C and the p adjusted to 5.0. 600 g/ton D.S. beta-amylase (Biozyme M II - Amano Pharmaceutical Co., Ltd, Nagoya, Japan, containing 33.4 beta-amylase units per g) and different amounts of debranching enzyme were added and the reaction mixtures sampled after 72 hours. The percent maltose was determined by HPLC. The following results were obtained: EMI34.1 <tb> Debrancing <SEP> % <SEP> % <SEP> <tb> enzyme <SEP> (PU/g <SEP> D.S.) <SEP> Dextrose <SEP> Maltose <tb> <SEP> O <SEP> (control) <SEP> 0.3 <SEP> 58.7 <SEP> <tb> <SEP> 0.1 <SEP> 0.2 <SEP> 63.0 <tb> <SEP> 0.5 <SEP> 0.4 <SEP> 71.8 <tb> <SEP> 1 <SEP> 0.4 <SEP> 75.8 <tb> <SEP> 2. <SEP> 0.5 <SEP> ?8.4 <SEP> <tb> <SEP> 4 <SEP> 0.4 <SEP> 79.6 <tb> These results indicate that when debranching enzyme is used in conbination with beta-amylase, considerably higher maltose levels can be obtained. Example 1t 114 g of Tapioca starch were slurried with 886 ml of deionized water to which had been added 0.5 g calcium chloride. -The slurry was cooked by heating to approximately 1000 C and then cooled to 500 C. The pH was adjusted to 5.7 and 0.S6gofBiozyme M II and an amount of debra.nching enzyme corresponding to 2 PU/g D.S. were added. The pH was kept constant at 5.5. -The reaction mixture was sampled and analyzed by HPLC. EMI35.1 <tb> Reaction <SEP> time <tb> <SEP> (hours) <SEP> Dextrose <SEP> Maltose <tb> <SEP> 24 <SEP> 0.5 <SEP> 77.4 <tb> <SEP> 48 <SEP> - <SEP> 0.6 <SEP> - <SEP> 84.4 <tb> This demonstrates that debranching enzyme can be used together with beta-amylase to produce extra-high maltose syrups.

Claims: CLAIMS 1. A debranching. enzyme product which comprises a novel debranching enzyme of the pullulanase type having the following characteristics: al it is obtainable from a fermentation broth produced by cultivating in a suitable nutri ent medium a strain of the taxonomic group herein named Bacillus acidopullulyticus, b) it exhibits enzyme chemical properties essentially identical to and immunological properties identical or partially identical to those of. the debranching enzyme derived from the Bacillus strain NCIB 11607, c) its activity optimum, measured by incubation for 30 minutes in acetate buffer (0.05 M) at pH 4 - 5 is at least about 600C, d) its pH optimum is in the range of 3.5 to 5.5 as determined by incubation for 30 minutes in acetate buffer (0.05 M) at about 60 C, and e) it has a residual activity after 72 hours at 60 0C as measured in a dextrose solution (30 percent D.S. by weight) at pH 5 of at least 50 percent. 2. The debranching enzyme product of claim 1 in which the debranching enzyme activity is in the range of from 10 to 350,000 pullulanase. units per g. 3. The debranching enzyme product of claim 2 in which the activity is in the range of from .100 to 15,000 pullulanase units per g. 4. The debranching enzyme product resulting from cultivation of a debranching enzyme producing strain of Bacillus acidoul1ulyticus in a suitable nutrient medium and recovery of the debranching enzyme from the culture containing medium. 5. A process for the preparation of the debranching enzyme product of claim 1, which prooess comprises the cultivation, in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts, of a debranching enzyme producing Bacillus strain belonging to the taxonomic group Bacillus acido- rxzlluivticus, followed by recovery of said debranching enzyme product. 6. The'process of claim 5, in which the Bacillus acidopullulvticus strain is selected from the group consisting of NCIB 11607, BCIB 11610, and variants or mutants thereof 7. The process of claim 6, in which the strain is selected from the mutant group consisting of NCIB 11638 and NCIB 11647. 8. The process of Claim 5, in which the Bacillus acidopullulyticus strain is NCIB 11611, our a variant or mutant thereof. 9. The process of Claim 5, in which the Bacillus acidopullulyticus strain is NCIB 116362 or a variant or mutant thereof. 10. The process of Claim 5, in which the Bacillus acidopullulyticus strain is NCIB 11637, or a variant or mutant thereof. 11. The process of Claim 5, in which the Bacillus aciaopullulyticus strain is NCIB 11639, or a variant or mutant thereof. 12. A process for converting starch into syrups containing dextrose and/or maltose, which process comprises conducting the saccharification of starch or starch hydrolysates in the presence of an enzyme system which comprises effective amounts of the novel debranching enzyme of Claim 4 and a saccharifying enzyme selected from the group consisting of glucoamylase and beta-amylase. 13. The process of Claim 12, further comprising saccharification of a starch hydrolysate of at least 30 percent by weight of dry solids. 14. The process of Claim 12 in which the saccharification is conducted in the pH-range of from 3.5 to 5.5 at a temperature in the range of from 550 to 650C. 15. -The process of claim 12 in which the dosages of glucoamylase and beta-amylase are in the range of from 0.05 to 0.5 AG units and from 0.005 to 0.3 beta-amylase units, respectively, per g of dry solids. 16. The process of claim 15 in which the dosage of debranching enzyme. is in the range of frctn 0.005 to 5 pullulanase units per g of dry solids.

Inventors: DIERS, IVAN VERNER, NIELSEN, GRETHE CAMILLA, NORMAN, BARRIE EDMUND, OUTTRUP, HELLE
Applicants: NOVO INDUSTRI A/S
Application year: 1982
Main classification: C12N9
IPC codes: C12P19, C12N9, C12R1