WO2012047430A1 - Processing of palm kernel waste using mannanase and pectinase - Google Patents

Processing of palm kernel waste using mannanase and pectinase Download PDF

Info

Publication number
WO2012047430A1
WO2012047430A1 PCT/US2011/050349 US2011050349W WO2012047430A1 WO 2012047430 A1 WO2012047430 A1 WO 2012047430A1 US 2011050349 W US2011050349 W US 2011050349W WO 2012047430 A1 WO2012047430 A1 WO 2012047430A1
Authority
WO
Grant status
Application
Patent type
Prior art keywords
mannanase
pectinase
amount
waste
palm kernel
Prior art date
Application number
PCT/US2011/050349
Other languages
French (fr)
Inventor
Christopher Barnett
Peter Nelson Birschbach
Original Assignee
Danisco Us Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/02Monosaccharides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/14Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Abstract

Described are compositions and methods for processing palm kernel waste (PKW) using a combination of mannanase and pectinase to significantly improve mannose yield.

Description

PROCESSING OF PALM KERNEL WASTE USING

MANNANASE AND PECTINASE

PRIORITY

[01] The present application claims priority to U.S. Provisional Application Serial No. 61 /390,846, filed October 7, 2010, which is incorporated by reference in its entirety.

TECHNICAL FIELD

[02] Described are compositions and methods for processing palm kernel waste (PKW) using a combination of mannanase and pectinase. BACKGROUND

[03] Palm kernel waste (PKW) is a by-product from the process of extracting palm oil from oil palm kernels. PKW is typically a ground or pulverized composition containing, e.g., palm kernel shells, mesocarp fibers, and empty fruit bunches. PKW can be used as biomass, either directly as a fuel source (i.e., to burn and produce energy) or via enzymatic processing to produce more environmentally friendly forms of fuel.

[04] PKW is rich in galacto-mannans (typically 30-50% by weight), and can be processed using mannanases to yield significant quantities of mannose. Mannose has a variety of uses, e.g., in the food and beverage industries, and represents a higher-value product than biomass.

SUMMARY

[05] Described are compositions and methods for preparing mannose from palm kernel waste (PKW), involving contacting the PKW with a combination of a mannanase and a pectinase.

[06] In one aspect, a method of preparing mannose from palm kernel waste is provided, comprising: contacting the palm kernel waste with a pectinase and a mannanase, wherein the amount of mannose produced by contacting the palm kernel waste with the pectinase and the mannanase is greater than the amount of mannose produced by contacting an equivalent amount of palm kernel waste with an equivalent amount of the mannanase in the absence of the pectinase.

[07] In some embodiments, the palm kernel waste is contacted with the pectinase and the mannanase in the same reaction vessel. In some embodiments, the palm kernel waste is contacted with the pectinase and the mannanase simultaneously. In some embodiments, the palm kernel waste is contacted with the pectinase and the mannanase sequentially. In some embodiments, the palm kernel waste is first contacted with the pectinase and then contacted with the mannanase.

[08] In some embodiments, the pectinase and the mannanase are present in an amount sufficient to produce a mannanase to pectinase activity ratio of from about 10,000:1 to about 200,000:1 , based on activity defined in U/g. In some embodiments, the pectinase and the mannanase are present in an amount sufficient to produce a mannanase to pectinase activity ratio of from about 35,000:1 to about 140,000:1 , based on activity defined in U/g.

[09] In some embodiments, the amount of pectinase is sufficient to produce a pectinase activity of from about 0.18 to about 3.6 U/g PKW. In some embodiments, the amount of mannanase is sufficient to produce a

mannanase activity of from about 12,250 to about 250,000 U/g PKW.

[10] In some embodiments, the pectinase is a plurality of pectinases. In some embodiments, the mannanase is a plurality of mannanases.

[11] In some embodiments, the amount of mannose produced by contacting the palm kernel waste with the pectinase and the mannanase is greater than the amount of mannose produced by contacting an equivalent amount of palm kernel waste with twice the equivalent amount of the mannanase in the absence of the pectinase.

[12] In some embodiments, the amount of mannose produced by contacting the palm kernel waste with the pectinase and the mannanase is twice the amount of mannose produced by contacting an equivalent amount of palm kernel waste with the equivalent amount of the mannanase in the absence of the pectinase. In some embodiments, the amount of mannose produced by contacting the palm kernel waste with the pectinase and the mannanase is three times the amount of mannose produced by contacting an equivalent amount of palm kernel waste with the equivalent amount of the mannanase in the absence of the pectinase.

[13] In another aspect, mannose produced by any of the described methods is provided.

[14] In another aspect, a composition for use in preparing mannose from palm kernel waste is provided, comprising: (a) a pectinase, and (b) a mannanase.

[15] In some embodiments, the pectinase and the mannanase are present in an amount sufficient to produce a mannanase to pectinase activity ratio of from about 10,000:1 to about 200,000:1 , based on activity defined in U/g. In some embodiments, the pectinase and the mannanase are present in an amount sufficient to produce a mannanase to pectinase activity ratio of from about 35,000:1 to about 140,000:1 , based on activity defined in U/g.

[16] In some embodiments, the amount of pectinase is sufficient to produce a pectinase activity of from about 0.18 to about 3.6 U/g PKW. In some embodiments, the amount of mannanase is sufficient to produce a

mannanase activity of from about 12,250 to about 250,000 U/g PKW.

[17] In some embodiments, the pectinase is a plurality of pectinases. In some embodiments, the mannanase is a plurality of mannanases

[18] These and other aspects and embodiments of the present

compositions and method will be apparent in view of the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

[19] Figure 1 is a graph showing the amount of mannose produced

(expressed as mg mannose/g PKW) from PKW treated with 1 .0% mannanase (first bar, labeled "A (2x)") or 0.5% mannanase plus 0.5% of a second indicated carbohydrase, (all subsequent bars). The reference codes (A-L) for Figure 1 are given in Table 1 . DETAILED DESCRIPTION

I. Overview

[20] Described are compositions and methods for preparing mannose from palm kernel waste (PKW), involving contacting the PKW with a combination of a mannanase and a pectinase. This combination of enzymes produces more mannose from PKW that is produced by either enzyme alone. In some cases, the increase in mannose yield that results from the use of pectinase is about 2-fold, or more. II. Definitions

[21] Prior to describing the present compositions and methods in detail, the following terms are defined for clarity. Terms not defined should be accorded their ordinary meanings as used in the relevant art.

[22] As used herein, a "pectinase" is an enzyme capable of hydrolyzing the substrates pectin, pectate, and/or derivatives, thereof, and/or is encompassed by the enzyme classifications EC 4.2.2.2, EC 4.2.2.10, or EC 4.2.2.22.

[23] As used herein, a "mannanase" is an enzyme capable of hydrolyzing the substrate mannan, and/or derivatives, thereof, and/or is encompassed by the enzyme classifications EC 3.2.1 .25, EC 3.2.1 .78, or EC 3.2.1 .1 1 3.

Preferred mannanases release free mannose, either alone or in combination with other enzymes.

[24] As used herein, "mannose" refers to a-D-mannopyranose, β-D- mannopyranose, a-D-mannofuranose, β-D-mannofuranose, and mixtures or blends, thereof, any one or more of which may, in various embodiments, be expressly included or excluded from the definition.

[25] As used herein, "pectinase activity" is defined in units/gram (U/g)), and may be determined using the following assay: A mixture of apples (50% Golden Delicious, 25% Cox Orange and 25% Lobo) are ground in a meat grinder, pressed and pasteurized, to obtain a juice having an outlet time of approximately 60 seconds (compared to 20 seconds for water). Cold juice (approximately 3°C) is pipetted into glass containers in about 25 ml portions and 1 ml enzyme solution (of known concentration) is added per glass and mixed to treat the juice. The juice and enzyme mixtures are incubated for about 1 hr at 50 °C. The concentration of enzyme solution should be adjusted so that juice treated with 100% enzyme solution has an outlet time of 1 -2 seconds more than a completely depectinised juice. There should also be at least a 5-second difference in the outlet time between juice treated with a 25% enzyme solution and a 100% enzyme solution. Following pasteurization and sifting through a nylon cloth, the viscosity of the treated juice is measured at 25 °C with a capillary viscosimeter. Viscosity (in seconds) is plotted as a function of the dilution factors 1 .00, 0.75, 0.50, and 0.25 (i.e., 100%, 75%, 50%, and 25% enzyme solution). The plotted data can be compared to a standard curve to determine pectinase activity in a sample. The exemplary pectinase composition (MULTIFECT® Pectinase FE) has known pectinase activity and can be used as a standard.

[26] As used herein, "mannanase activity" is defined in units/gram (U/g), as determined using the following assay: 20 g 3,5-dinitrosalicylic acid (DNS) is added to 1 L deionized water in a 2 L beaker. 300 ml 10.67% sodium hydroxide is added and the suspension is heated on a stir plate (not to exceed 50°C) until clear. 600 g potassium sodium tartrate tetrahydrate is gradually added to the solution with continual mixing and the solution is allowed to reach room temperature, diluted to 2 L, and optionally filtered through a course sintered glass filter. The solution is stored in a dark amber bottle at room temperature.

[27] 1 .4 g locust bean gum is gradually added to 500 ml Tris-HCI buffer (15.67 g Tris-HCI adjusted to pH 7.5 with ammonium hydroxide (1 .5%) in a 2 L volume) in a 1 L beaker at 60°C for 60 minutes, and then cooled to room temperature and adjusted to 500 ml with deionized water. Cleared

supernatant (subjected to centrifugation at 3,500 rpm for 10 minutes) is used the as a substrate for a known amount (i.e., g or g/L) of mannanase standard and unknown mannanase sample. The amount of standard and sample used are selected to be within the linear range of the assay, where the change in absorbance (ΔΑ) is from about 0.1 7 to about 0.52. Standard and sample concentrations between 0.050-0.140 mannanase units/L typically fall within the linear range of this assay. Where necessary, samples can be diluted in the Tris-HCI buffer.

[28] To perform the assay, 2 ml locust bean gum substrate is equilibrated in 16x100 mm glass test tubes for 20 minutes at 40 °C. 0.5 ml of enzyme sample dilution is added, mixed, and incubate for 10 minutes. The reaction is stopped by the addition of 3.0 ml of the DNS-solution, mixed, boiled for 15 min in a covered test tube to prevent evaporation, cooled in an ice water bath for 50 min, and allowed to equilibrate to room temperature for 10 minutes prior to reading the absorbance at 540 nm. The measurement is compared to a deionized water blank.

[29] The reaction control (i.e., reagent/enzyme blank) is the amount of reducing sugars present in the locust bean gum substrate and/or present in the enzyme sample. As before, 2 ml locust bean gum substrate is

equilibrated in 16x1 00 mm glass test tubes for 20 minutes at 40 °C. However, 3.0 ml of DNS-solution is added and mixed prior to the addition of 0.5 ml of enzyme dilution, and then boiled, cooled, equilibrated, and read at 540 nm against a deionized water blank.

[30] ΔΑ is determined by subtracting the average absorbance values for the reagent/enzyme blanks from the absorbance readings of reactions in which enzyme is present. A standard curve is prepared using linear regression, where net absorbance is plotted on the y-axis and concentration (mannanase units/liter) is plotted on the x-axis, and the mannanase activity of each sample is determined based on the standard curve. The exemplary mannanase composition (GC266) has known mannanase activity and can be used as a standard.

[31] As used herein, "palm kernel waste (PKW)" refers to by-products from the process of extracting palm oil or other materials from oil palm kernels. PKW may include palm kernel shells, mesocarp fibers, empty fruit bunches, and/or other materials. PKW may be in a ground or pulverized form.

[32] As used herein, the term "contacting" refers to bringing specified components, e.g., an enzyme and a substrate, into physical contact.

Contacting includes mixing dry compositions and mixing liquid compositions, or combinations, thereof.

[33] As used herein, the term "equivalent amount," with reference to a substrate, enzyme, or other specified component, refers to the same or equal amount (in terms of, e.g., units, grams, or moles) with reference to an antecedent composition and amount. For example, with reference to a composition comprising 10 U of mannanase activity and 20 U of pectinase activity, a composition having an equivalent amount of mannanase activity has 10 U of mannanase activity.

[34] As used herein, the phrase "substantially free of an activity" (or similar phrases) means that a specified activity is either undetectable in an admixture of polypeptides, or present in an amount that would not interfere with the intended purpose of the admixture.

[35] As used herein, the singular articles "a," "an," and "the" encompass the plural referents unless the context clearly dictates otherwise.

[36] The following abbreviations/acronyms have the following meanings unless otherwise specified:

Figure imgf000009_0001

[37] All references cited herein are hereby incorporated by reference in their entirety. III. Compositions and methods for preparing mannose from PKW

[38] Described are compositions and methods for preparing mannose from palm kernel waste (PKW), involving contacting the PKW with a combination of a mannanase and a pectinase. The combination of these two enzymes results in a significantly higher yield of mannose that can be obtained using either enzyme alone.

[39] Preferably, PKW is contacted with the mannanase and the pectinase in the same reaction vessel, avoiding the need to transfer PKW to different reaction vessels for different portions of the enzymatic treatment. In some cases, PKW is contacted with the mannanase and the pectinase

simultaneously. In other cases, the PKC is contacted with the mannanase and the pectinase sequentially. Where PKC is contacted with the mannanase and the pectinase sequentially, PKC is preferably first contacted with the pectinase and then the mannanase, although PKC may also be contacted first with the mannanase and then with the pectinase. Where mannanase and pectinase are contacted with PKW simultaneously, they are ideally included together in a single composition.

[40] Examples of pectinases suitable for use as described include enzymes encompassed by the enzyme classifications EC 4.2.2.2 (i.e., pectate lyase enzymes that favors pectate, the anion, over pectin, the methyl ester); EC 4.2.2.10 (i.e., pectin lyase enzymes that favors pectin, the methyl ester, over pectate, the anion); and EC 4.2.2.22 (i.e., pectate trisaccharide-lyase or exopectate-lyase).

[41] In some embodiments, the pectinase is derived from a fungal organism, such as an Aspergillus spp., a Penicillium spp., or a Trichoderma spp. In some embodiments, the pectinase is derived from a bacterium, such as an Erwinia spp., a Pseudomonas spp., a Klebsiella spp., a Xanthomonas spp., a Bacillus spp. (e.g., Nasser et al. (1993) FEBS Letts. 335:319-26; Kim ef al. (1994) Biosci. Biotech. Biochem. 58:947-49; Dave and Vaughn (1 971 ) J. Bacterid. 108:166-74; Nagel and Vaughn (1961 ) Arch. Biochem. Biophys. 93:344-52; Karbassi and Vaughn (1980) Can. J. Microbiol. 26:377-84, Hasegawa and Nagel (1966) J. Food Sci. 31 :838-45; and Kelly and Fogarty (1978) Can. J. Microbiol. 24:1 164-72). Particular pectinases can be obtained from Bacillus subtilis.

[42] Suitable pectinases may be divalent cation-independent and/or thermostable. In particular embodiments, the pectinase is as described in Heffron ef al. (1995) Mol. Plant-Microbe Interact. 8:331 -34; Henrissat ef al. (1995) Plant Physiol. 107: 963-76; WO 99/27083; WO 99/27084; WO

02/006442; or U.S. Pat. No. 6,284,524.

[43] Specific examples of suitable commercially available pectinase products include CELLULOSIN™ PC5, PE60, PEL, and ME (HBI Products), SUMIZYME™ AP2, PX, PMAC, PCLA, MC, and SPG (Shin Nihon), and

MULTIFECT® Pectinase FE (Genencor), each of which includes a pectinase from Aspergillus niger, PECTINASE™ G, GL, and PL (Amano), which includes a pectinase(s) from Aspergillus niger and/or Apregillus pulverulentes, PECTINEX™ (Novozymes), which includes a pectinase from Aspergillus niger and/or Aspergillus aculeatus, PRIMAGREEN® EcoScour (Genencor), SCOURZYME™ (Novozymes), and PECTINASE™ XP-534 (Nagase

ChemteX), each of which includes a pectinase from Bacillus, as well as GAMMAPECT™ PCL and ROHAPECT® (AB Enzymes), RAPIDASE® X- Press and C80L (DSM Food specialties), SUKULASE™ N and S (Sankyo Lifetech), PECTINASE-GODO™ (Godo Syusei), and BIOPREP™,

CITRAZYME™ and VINOZYME™ (Novozymes).

[44] In some embodiments, a single pectinase is used. In some

embodiments, a plurality of pectinases is used.

[45] Examples of mannanases suitable for use as described include enzymes encompassed by the enzyme classifications EC 3.2.1 .25 (i.e., mannase; β-D-mannosidase; β-mannoside mannohydrolase; exo-β-ϋ- mannanase; or β-D-mannoside mannohydrolase); EC 3.2.1 .78 (i.e., endo-1 ,4- β-mannanase; β-mannanase); and EC 3.2.1 .1 13 (i.e., mannosidase; 1 ,2-a- mannosidase; exo-a-1 ,2-mannanase; mannose-9 processing a- mannosidase).

[46] The mannanase enzymes may be of bacterial or fungal origin. In some embodiments, the mannanase is derived from a strain of filamentous fungus, such as an Aspergillus spp. (WO 94/25576) or Trichoderma spp. (e.g., WO 93/24622). In some embodiments, the mannanase is derived from a bacterium, such as a Bacillus spp. (e.g., Talbot et al. (1990) Appl. Environ. Microbiol. 56:3505-10; Mendoza et al. (1994) World J. Microbiol. Biotech. 10:551 -55; JP-A-03047076; JP- A-63056289; JP-A- 63036775; JP-A- 08051975; WO 97/1 1 164; or WO 99/64619) or a Humicola spp. (e.g., WO 99/64619).

[47] Specific examples of suitable commercially available mannanase products include MANNASTAR® 375 (Genencor), GC 266 (Genencor), ECONASE® MP 1 000 (AB Enzymes), and ROHALASE® GMP (AB

Enzymes), each of which includes a mannanase from Trichoderma reesei, CELLULOSINTM GM5 (HBI Products) and SUMIZYME™ ACH (Shin Nihon), each of which includes a mannanases from Aspergillus niger, and

MANNAWAY® (Novozymes).

[48] In some embodiments, a single mannanase is used. In some embodiments, a plurality of mannanases is used.

[49] On an activity basis, varying amounts of pectinase and mannanase enzymes can be used, where pectinase and mannanase activity can be measured as described, herein. In some embodiments, the amount of pectinase used is at least about 0.1 pectinase units per gram of palm kernel extruder waste (U/g PKW). In some embodiments, the amount of pectinase used is at least about 0.1 , at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1 .0, at least about 1 .1 , at least about 1 .2, at least about 1 .3, at least about 1 .4, at least about 1 .8, at least about 1 .9, or even at least about 2 U/g PKW. Exemplary ranges are from about 0.18 to about 3.6, from about 0.36 to about 3.24, from about 0.54 to about 3.06, from about 0.72 to about 2.88, from about 0.90 to about 2.70, and from about 0.90 to about 1 .80 U/g PKW.

[50] In some embodiments, the amount of mannanase used is at least about 12,000 mannanase units per gram of palm kernel extruder waste (U/g PKW). In some embodiments, the amount of mannanase used is at least about 21 ,250, at least about 25,000, at least about 41 ,250, at least about 50,000, at least about 62,500, at least about 75,000 U/g PKW. Exemplary ranges are from about 12,250 to about 250,000, from about 25,000 to about 225,000, from about 35,000:1 to about 140,000:1 , from about 37,500 to about 212,500, from about 41 ,250 to about 212,500, from about 50,000 to about 200,000, from about 62,500 to about 187,500, and from about 62,500 to about 125,000 U/g PKW.

[51] On an activity ratio basis, varying ratios of pectinase and mannanase enzymes can be used as described. In some embodiments, the ratio of mannanase activity to pectinase activity (each in U/g PKW) is from about 10,000:1 to about 200,000:1 , from about 20,000:1 to about 150,000:1 , from about 30,000:1 to about 120,000:1 , or even from about 60,000:1 to about 70,000:1 .

[52] The temperature and time of incubation of the mannanase and pectinase with the PKW is not believed to be critical, long as the conditions do not prematurely inactivate one or both of the enzymes. In some

embodiments, the time of incubation using mannanase and pectinase, together, is less the time of incubation using either mannanase or pectinase, alone. The selection of particular incubation temperatures and times of incubation depend on the particular mannanase and pectinase used and the desired results. Lower temperatures (i.e., close to ambient temperature) are generally preferred for cost and environmental reasons, while elevated temperatures (i.e., significantly above ambient temperature) are generally preferred to increase enzyme activity. Shorter incubation times are generally preferred to increase throughput, although overnight or longer incubations may allow the use of less enzyme.

[53] Compositions containing mannanase and/or pectinase may further include any number of buffers, salts, stabilizing agents, formulation agents, surfactants, polymers, dyes, or additional enzymes. Exemplary additional enzymes include but are not limited to cellulases, xylanases, amylases, and proteases. However, in some embodiments, mannanase and/or pectinase compositions are substantially free of other enzymatic activities, such as cellulase, xylanase, amylase, and protease activities.

[54] These and other features of the present compositions and methods will be apparent from the description and appended Examples. EXAMPLES

[55] An initial screen of mannanase in combination with selected carbohydrase enzymes was performed to identify enzyme combinations that produced a beneficial effect.

[56] Briefly, a series of palm kernel waste (PKW) samples was prepared by adding 15 mL of 50 mM sodium citrate (pH 3.5-4.5) to 15 grams of PKW. 1 .0% mannanase product (w/w) or 0.5% mannanase product plus 0.5% of a second carbohydrase (w/w) (or no second carbohydrase, as a control) was then added to each sample, followed by incubation at 55°C for 23 hours. The identity of the enzymes tested is shown in Table 1 , wherein "ID" corresponds to the identifier used in the graph. The first bar of the graph represents a double dose (1 %) of mannanase (i.e. component "A"), while the all subsequent bars represent 0.5% mannanase ("A") plus the indicated second carbohydrase ("B" through "L" or no second carbohydrase ("none").

Table 1. Enzymes tested.

Figure imgf000015_0001

[57] The particular mannanase composition (GC 266) used had a minimum activity of 12,500,000 mannanase units/gram, as measured by mannanase assay described, herein. 1 .0% mannanase is equivalent to 125,000 mannanase units per gram of palm kernel extruder waste, and 0.5% mannanase is equivalent to 62,500 mannanase units per gram of palm kernel extruder waste. The level of pectinase activity in the mannanase preparation was assumed to be nominal.

[58] The particular pectinase composition (MULTIFECT® Pectinase FE) used had a minimum activity of 180 pectinase units/gram, as measured pectinase assay described, herein. 1 .0% pectinase is equivalent to 1 .8 pectinase units per gram of palm kernel extruder waste and 0.5% pectinase is equivalent to 0.9 pectinase units per gram of palm kernel extruder waste. The level of mannanase activity in the pectinase preparation was assumed to be nominal. [59] Following incubation, the samples were diluted to 100 grams nominal weight by the addition of 70 grams water. The samples were mixed thoroughly and a small aliquot was removed and subjected to centrifugation at 13,000 x g.

[60] The supernatants were loaded onto an Agilent 1 100 series HPLC (Santa Clara, CA, USA), and separated using a Bio-Rad Aminex HPX-87C (Hercules, CA, USA) column at 80°C. Mannose peaks were detected based on refractive index, and the peaks were integrated using full baseline. The resulting mannose concentration was calculated using linear regression based on a mannose standard curve prepared using 1 g/L, 5 g/L and 10 g/L mannose.

[61] As shown in Figure 1 , of the combinations tested, only mannanase combined with a pectinase (labeled "F"), or mannanase combined with all the tested carbohydrases including the pectinase (labeled "ALL"), demonstrated increased mannose yield, even compared to twice the dose of mannanase alone [labeled "A (2X)"].

[62] In a further effort to optimize the amount of mannanase required to maximize mannanase yield, the mannanase:pectinase ratio was varied and a similar experiment was performed. The results are shown in Table 2.

Table 2. Mannose yields using different amounts of mannanase

and pectinase.

Figure imgf000016_0001

[63] As shown in the table, the best mannose yields (i.e., about 8.9% release) were obtained using about 0.5% mannanase and about 0.5% pectinase. By comparison, using mannanase alone resulted in a 2.6% release and using pectinase alone resulted in a 2.0% release. The increase in mannose release obtained using the combination of mannanase and pectinase was, therefore, greater than 3-fold compared to either mannanase or pectinase, alone.

[64] Different levels of mannose can be released using different ratios and amount of mannanase and pectinase, depending on the desired results.

Claims

CLAIMS What is claimed is:
1 . A method of preparing mannose from palm kernel waste, comprising: contacting the palm kernel waste with a pectinase and a mannanase, wherein the amount of mannose produced by contacting the palm kernel waste with the pectinase and the mannanase is greater than the amount of mannose produced by contacting an equivalent amount of palm kernel waste with an equivalent amount of the mannanase in the absence of the pectinase.
2. The method of claim 1 , wherein the palm kernel waste is contacted with the pectinase and the mannanase in the same reaction vessel.
3. The method of claim 1 , wherein the palm kernel waste is contacted with the pectinase and the mannanase simultaneously.
4. The method of claim 1 , wherein the palm kernel waste is contacted with the pectinase and the mannanase sequentially.
5. The method of claim 4, wherein the palm kernel waste is first contacted with the pectinase and then contacted with the mannanase.
6. The method of any of the preceding claims, wherein the pectinase and the mannanase are present in an amount sufficient to produce a mannanase to pectinase activity ratio of from about 10,000:1 to about 200,000:1 , based on activity defined in U/g.
7. The method of any of the preceding claims, wherein the pectinase and the mannanase are present in an amount sufficient to produce a mannanase to pectinase activity ratio of from about 35,000:1 to about 140,000:1 , based on activity defined in U/g.
8. The method of any of the preceding claims, wherein the amount of pectinase is sufficient to produce a pectinase activity of from about 0.18 to about 3.6 U/g PKW.
9. The method of any of the preceding claims, wherein the amount of mannanase is sufficient to produce a mannanase activity of from about 12,250 to about 250,000 U/g PKW.
10. The method of any of the preceding claims, wherein the pectinase is a plurality of pectinases.
1 1 . The method of any of the preceding claims, wherein the mannanase is a plurality of mannanases.
12. The method of any of the preceding claims, wherein the amount of mannose produced by contacting the palm kernel waste with the pectinase and the mannanase is greater than the amount of mannose produced by contacting an equivalent amount of palm kernel waste with twice the equivalent amount of the mannanase in the absence of the pectinase.
13. The method of any of the preceding claims, wherein the amount of mannose produced by contacting the palm kernel waste with the pectinase and the mannanase is twice the amount of mannose produced by contacting an equivalent amount of palm kernel waste with the equivalent amount of the mannanase in the absence of the pectinase.
14. The method of any of the preceding claims, wherein the amount of mannose produced by contacting the palm kernel waste with the pectinase and the mannanase is three times the amount of mannose produced by contacting an equivalent amount of palm kernel waste with the equivalent amount of the mannanase in the absence of the pectinase.
15. Mannose produced by the method of any of the preceding claims.
16. A composition for use in preparing mannose from palm kernel waste, comprising:
(a) a pectinase, and
(b) a mannanase.
17. The composition of claim 16, wherein the pectinase and the mannanase are present in an amount sufficient to produce a mannanase to pectinase activity ratio of from about 10,000:1 to about 200,000:1 , based on activity defined in U/g.
18. The composition of claim 16 or 17, wherein the pectinase and the mannanase are present in an amount sufficient to produce a mannanase to pectinase activity ratio of from about 35,000:1 to about 140,000:1 , based on activity defined in U/g.
19. The composition of any of claims 16-18, wherein the amount of pectinase is sufficient to produce a pectinase activity of from about 0.18 to about 3.6 U/g PKW.
20. The composition of any of claims 16-19, wherein the amount of mannanase is sufficient to produce a mannanase activity of from about 12,250 to about 250,000 U/g PKW.
21 . The composition of any of claims 16-20, wherein the pectinase is a plurality of pectinases.
22. The composition of any of claims 16-21 , wherein the mannanase is a plurality of mannanases.
PCT/US2011/050349 2010-10-07 2011-09-02 Processing of palm kernel waste using mannanase and pectinase WO2012047430A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US39084610 true 2010-10-07 2010-10-07
US61/390,846 2010-10-07

Publications (1)

Publication Number Publication Date
WO2012047430A1 true true WO2012047430A1 (en) 2012-04-12

Family

ID=44678037

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/050349 WO2012047430A1 (en) 2010-10-07 2011-09-02 Processing of palm kernel waste using mannanase and pectinase

Country Status (1)

Country Link
WO (1) WO2012047430A1 (en)

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6336775A (en) 1986-07-31 1988-02-17 Toshiro Akino Novel alkalophilic strain of bacillus genus capable of producing beta-mannanase and beta-mannosidase and use thereof
JPS6356289A (en) 1986-07-30 1988-03-10 Toshiro Akino Beta-mannanase and production thereof
JPH0347076A (en) 1989-08-25 1991-02-28 Toshiro Akino Beta-mannase and production thereof
WO1993024622A1 (en) 1992-05-22 1993-12-09 Alko Ltd. Mannanase enzymes, genes coding for them, methods for isolating the genes, and methods for bleaching lignocellulosic pulps
WO1994025576A1 (en) 1993-04-30 1994-11-10 Novo Nordisk A/S An enzyme exhibiting mannanase activity
JPH0851975A (en) 1991-10-09 1996-02-27 Godo Shiyusei Kk New beta-mannanase and method for producing the same
WO1997011164A1 (en) 1995-09-20 1997-03-27 Genencor International, Inc. Purified mannanase from bacillus amyloliquefaciens and method of preparation
WO1999027083A1 (en) 1997-11-24 1999-06-03 Novo Nordisk A/S PECTIN DEGRADING ENZYMES FROM $i(BACILLUS LICHENIFORMIS)
WO1999027084A1 (en) 1997-11-24 1999-06-03 Novo Nordisk A/S Novel pectate lyases
WO1999064619A2 (en) 1998-06-10 1999-12-16 Novozymes A/S Novel mannanases
US6284524B1 (en) 1997-11-24 2001-09-04 Novozymes A/S Pectate lyases
WO2002006442A2 (en) 2000-07-19 2002-01-24 Novozymes A/S Cell-wall degrading enzyme variants
CA2591650A1 (en) * 2006-06-15 2007-12-15 Grober Inc. Food products
WO2008113585A1 (en) * 2007-03-19 2008-09-25 Süd-Chemie AG Generation of chemical building blocks from plant biomass by selective depolymerization
WO2009074685A1 (en) * 2007-12-12 2009-06-18 Novozymes A/S Enzymatic degradation of biomass substrates comprising mannan
WO2010000858A1 (en) * 2008-07-03 2010-01-07 Novozymes A/S A personal wash bar

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6356289A (en) 1986-07-30 1988-03-10 Toshiro Akino Beta-mannanase and production thereof
JPS6336775A (en) 1986-07-31 1988-02-17 Toshiro Akino Novel alkalophilic strain of bacillus genus capable of producing beta-mannanase and beta-mannosidase and use thereof
JPH0347076A (en) 1989-08-25 1991-02-28 Toshiro Akino Beta-mannase and production thereof
JPH0851975A (en) 1991-10-09 1996-02-27 Godo Shiyusei Kk New beta-mannanase and method for producing the same
WO1993024622A1 (en) 1992-05-22 1993-12-09 Alko Ltd. Mannanase enzymes, genes coding for them, methods for isolating the genes, and methods for bleaching lignocellulosic pulps
WO1994025576A1 (en) 1993-04-30 1994-11-10 Novo Nordisk A/S An enzyme exhibiting mannanase activity
WO1997011164A1 (en) 1995-09-20 1997-03-27 Genencor International, Inc. Purified mannanase from bacillus amyloliquefaciens and method of preparation
US6284524B1 (en) 1997-11-24 2001-09-04 Novozymes A/S Pectate lyases
WO1999027083A1 (en) 1997-11-24 1999-06-03 Novo Nordisk A/S PECTIN DEGRADING ENZYMES FROM $i(BACILLUS LICHENIFORMIS)
WO1999027084A1 (en) 1997-11-24 1999-06-03 Novo Nordisk A/S Novel pectate lyases
WO1999064619A2 (en) 1998-06-10 1999-12-16 Novozymes A/S Novel mannanases
WO2002006442A2 (en) 2000-07-19 2002-01-24 Novozymes A/S Cell-wall degrading enzyme variants
CA2591650A1 (en) * 2006-06-15 2007-12-15 Grober Inc. Food products
WO2008113585A1 (en) * 2007-03-19 2008-09-25 Süd-Chemie AG Generation of chemical building blocks from plant biomass by selective depolymerization
WO2009074685A1 (en) * 2007-12-12 2009-06-18 Novozymes A/S Enzymatic degradation of biomass substrates comprising mannan
WO2010000858A1 (en) * 2008-07-03 2010-01-07 Novozymes A/S A personal wash bar

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
DAVE, VAUGHN, J. BACTERIOL., vol. 108, 1971, pages 166 - 74
HASEGAWA, NAGEL, J. FOOD SCI., vol. 31, 1966, pages 838 - 45
HEFFRON ET AL., MOL. PLANT-MICROBE INTERACT., vol. 8, 1995, pages 331 - 34
HENRISSAT ET AL., PLANT PHYSIOL., vol. 107, 1995, pages 963 - 76
KARBASSI, VAUGHN, CAN. J. MICROBIOL., vol. 26, 1980, pages 377 - 84
KELLY, FOGARTY, CAN. J. MICROBIOL., vol. 24, 1978, pages 1164 - 72
KIM ET AL., BIOSCI. BIOTECH. BIOCHEM., vol. 58, 1994, pages 947 - 49
MENDOZA ET AL., WORLD J. MICROBIOL. BIOTECH., vol. 10, 1994, pages 551 - 55
NAGEL, VAUGHN, ARCH. BIOCHEM. BIOPHYS., vol. 93, 1961, pages 344 - 52
NASSER ET AL., FEBS LETTS., vol. 335, 1993, pages 319 - 26
TALBOT ET AL., APPL. ENVIRON. MICROBIOL., vol. 56, 1990, pages 3505 - 10

Similar Documents

Publication Publication Date Title
Bhatia et al. Microbial β-glucosidases: cloning, properties, and applications
Topakas et al. Microbial production, characterization and applications of feruloyl esterases
Botella et al. Hydrolytic enzyme production by Aspergillus awamori on grape pomace
Aguilar et al. Microbial tannases: advances and perspectives
Bhargav et al. Solid-state fermentation: an overview
Chauhan et al. Mannanases: microbial sources, production, properties and potential biotechnological applications
Fazary et al. Feruloyl esterases as biotechnological tools: current and future perspectives
Chi et al. Bioproducts from Aureobasidium pullulans, a biotechnologically important yeast
Silva et al. Production of pectinase by solid-state fermentation with Penicillium viridicatum RFC3
Tolan et al. Cellulase from submerged fermentation
Motta et al. A review of xylanase production by the fermentation of xylan: classification, characterization and applications
Dhawan et al. Microbial mannanases: an overview of production and applications
Knob et al. β-Xylosidases from filamentous fungi: an overview
Sanghi et al. Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation
Numan et al. α-L-Arabinofuranosidases: the potential applications in biotechnology
Rodríguez Couto Exploitation of biological wastes for the production of value‐added products under solid‐state fermentation conditions
Sørensen et al. Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue
Ghose Measurement of cellulase activities
Sørensen et al. Enzymatic hydrolysis of wheat arabinoxylan by a recombinant “minimal” enzyme cocktail containing β‐xylosidase and novel endo‐1, 4‐β‐xylanase and α‐L‐arabinofuranosidase activities
Dhillon et al. Perspective of apple processing wastes as low-cost substrates for bioproduction of high value products: a review
WO2007109441A2 (en) Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2012125925A2 (en) Method for reducing viscosity in saccharification process
Iqbal et al. Purification and characterization of the kinetic parameters of cellulase produced from wheat straw by Trichoderma viride under SSF and its detergent compatibility
Ribeiro et al. Enzymes in juice processing: a review
WO1999027083A1 (en) PECTIN DEGRADING ENZYMES FROM $i(BACILLUS LICHENIFORMIS)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11761189

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11761189

Country of ref document: EP

Kind code of ref document: A1