WO2014149985A1

WO2014149985A1 - Cellulase and phytase inhibitor

Info

Publication number: WO2014149985A1
Application number: PCT/US2014/021734
Authority: WO
Inventors: Cristina POP; Yi Wu
Original assignee: Verenium Corp
Current assignee: BASF Enzymes LLC
Priority date: 2013-03-15
Filing date: 2014-03-07
Publication date: 2014-09-25
Anticipated expiration: 2015-09-15
Also published as: GB2512148A; GB201309574D0

Abstract

At acidic and neutral pH, Pemulen 1622 inhibited Cellulase and Phytase from hydrolyze their substrates, beta-glucan and phytic acid, respectively, without interfering with the spectroscopic method of detection. Previously, the mechanism of inhibition has been unknown, but experiments suggested that Pemulen 1622 decreased the concentration of available substrate or it bound competitively to the enzyme active site.

Description

CELLULASE AND PHYTASE INHIBITOR TECHNICAL FIELD

Natural and synthetic inhibitors of enzymes have been previously described especially in the pharmaceutical industry; however, enzyme inhibitors for cellulase, phytase, xylanase, and amylase enzymes are generally scarce.

Cellulase, phytase, xylanase, and amylase enzymes have a variety of known industrial uses including, but not limited to: oil well fracturing, animal feed additives, detergents, baking, manufacturing biofuels, etc. One of the challenges of using enzymes in these harsh industrial processes is to ensure that the enzyme is active at the right time under the optimal conditions, such as temperature or pH, for the enzymes characteristics. Therefore, a need exists to find an inhibitor for cellulase, phytase, xylanase, and amylase enzymes that will work under industrial conditions, which are much different than conditions required for the pharmaceutical industry.

Generally, it is understood in the art that enzymes can be inhibited by the binding of specific small molecules and ions. Enzyme inhibition can be reversible or irreversible. Irreversible inhibitors dissociates relatively slowly from the enzyme because the inhibitor has become either covalently or non-covalently bond to the enzyme. Reversible inhibition dissociates relatively quickly from the enzyme-inhibitor complex. Reversible inhibition can be competitive inhibition, non-competitive inhibition, or mixed inhibition. In competitive inhibition, the enzyme can bind substrate or the enzyme can bind with an inhibitor, but the enzyme cannot bind with both the substrate and the inhibitor. In non-competitive inhibition, the inhibitor and the substrate can bind to the enzyme molecule at different binding sites. Non-competitive inhibition cannot be overcome by increasing the substrate concentration. In mixed inhibition, a single inhibitor both hinders the binding of substrate and decreases the turnover number of the enzyme.

Previously, it has been shown in vitro that starch-g-poly(acrylic acid) copolymers and starch/poly (acrylic acid) mixtures have an inhibition potency for trypsin, which plays a key role in initiating the degradation of orally administered peptide drugs and in activating the zymogen forms of a lot of pancreatic peptidases. See, Ameye, et. al. "Trypsin inhibition, calcium and zinc ion binding of starch-g-poly(acrylic acid) copolymers and starch/poly(acrylic acid) mixtures for peroral peptide drug deliver" Journal of Controlled Release 75 (2001) 357-364. In another study, Carbopol 934, which is a cross-linked polyacrylic acid, was shown to inhibit R A-dependent DNA polymerase (reverse transcriptase) of Rauscher Murine Leukemia Virus (R-MuLV) and Avian Myoblastosis Virus (AMV); however, stimulation of enzymatic activity by Carbopol is not a property shared by all reverse transcriptases. See, Bloemers, "Inhibition of RNA-dependent DNA polymerase of OncoRNA viruses by Carbopol 934.

Although, it has been previously reported that a cross-linked polyacrylic acid, such as Carbopol can be an inhibitor of some proteases, and DNA polymerases, it has not been previously reported or suggested that a cross-linked copolymer of acrylic acid and C10-C30 alkyl acrylate, such as PEMULEN Polymers from Lubrizol Corporation, inhibit a cellulase, phytase, xylanase, or amylase enzymes.

As shown herein, using a cross-linked copolymers for inhibition of a cellulase, phytase, xylanase, or amylase enzymes has several advantages: 1) it is a reversible inhibitor, allowing activity recovery upon inhibitor dilution or dialysis; 2) it is a potent inhibitor, requiring small dosage for complete inhibition; 3) it seems to provide specific inhibition of cellulases or phytases, but not amylases and xylanases; 4) it is an emulsion stabilizer, and therefore it can serve a dual role when used in an emulsion formulation for controlled release, i.e., stabilizing the emulsion system, and further delaying the enzyme activity in addition to the encapsulation effect from the emulsion; 5) it may also be used in combination with other controlled release formulations, e.g., microencapsulation, nanoencapsulation, to improve the effect of delaying enzyme activity; this is based on the requirement of the dilution of the polymer upon enzyme release that needs to reach below the inhibition threshold. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

Figure 1 : shows that Pemulen is a potent inhibitor of Cellulase and Phytase.

Figure 2A-2D: show that Pemulen 1622 does not influence the standard curves of detection method below 1000 ug/mL.

Figure 3A-3B: shows that Pemulen 1622 is a reversible inhibitor of Phytase and

Cellulase.

Figure 4: shows that Pemulenl621 is a reversible potent inhibitor of Cellulase.

Figure 5 : shows that Aculin22 and Carbopol ETD 2623 also inhibit Cellulase.

Figure 6: shows Pemulen inhibition of Cellulase at pH 5-9. DETAILED DESCRIPTION OF THE INVENTION

"Cellulase" is an enzyme that catalyzes a reaction of endo-hydrolysis of 1,4-beta-D- glycosidic linkages in cellulose, lichenin, and cereal beta-D-glucans (such as barley beta-glucan). Since the predominant activities of the disclosed cellulase of the present invention are the endo- hydrolysis of barley beta-glucan and carboxymethyl cellulose, it is appropriately ascribed the IUBMB Enzyme Nomenclature EC 3.2.1.4. Other names used for enzymes belonging to this group include: endoglucanase, endo-l,4-beta-glucanase, carboxymethyl cellulase, and beta- 1,4- glucanase.

In an embodiment, the cellulase can be a cellulase or a variant of a cellulase disclosed in patent number: US 5,962,258, US 6,008,032, US 6,245,547, US 7,807,433, or international patent publication number WO 2009/020459. In another embodiment the cellulase can be a commercially available product including, but not limited to PYROLASE cellulase (Verenium).

"Phytase" is a phosphoric monoester hydrolase enzyme that catalyzes hydrolysis of phytic acid (myo-inositol-hexakisphosphate) to phosphorus and inositol. According to the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) and Bairoch A., "The ENZYME database in 2000," Nucleic Acids Res 28:304-305(2000), a phytase is classified as Enzyme Commission (EC) number EC 3.1.3.8, and is also referred to as: 1-phytase; myo-inositol-hexakisphosphate 3- phosphohydrolase; phytate 1 -phosphatase; phytate 3 -phosphatase; or phytate 6-phosphatase. Phytase is also classified as EC 3.1.3.26, which is also referred to as: 4-phytase; 6-phytase (name based on lL-numbering system and not ID-numbering); or phytate 6-phosphatase. Phytase is also classified as EC 3.1.3.72, which is also referred to as 5-phytase. Phytase is also referred to as histidine acid phosphatases (HAP); β-propeller phytases; purple acid phosphatase (PAP); and protein tyrosine phosphatases (PTPs). Alternative names for phytase will be known to those skilled in the art.

In an embodiment, the phytase is a phytase, or a phytase variant disclosed in International PCT Publication Number: WO 1999/008539, WO 2000/071728, WO 2001/090333, WO 2002/095003, WO 2006/028684, WO 2008/036916, or WO 2010/135588.

In another embodiment, the phytase is a commercially available phytase including but not limited to: PHYZYME (Dupont, Danisco, Genencor); QUANTUM and FINASE (AB Vista, AB Enzymes); NATUPHOS (BASF); RONOZYME (DSM); BIOFEED PHYTASE (Novo Nordisk); ALLZYME PHYTASE (Alltech); OPTIPHOS (Enzyvia, Phytex, Cornell); ROVABIO (Adisseo); and PHYTOUT (US Waters).

"Xylanase" (endo-1, 4-beta-xylanase, EC 3.2.1.8) is an enzyme that hydrolyze internal β- 1 ,4-xylosidic linkages in xylan to produce smaller molecular weight xylose and xylo-oligomers. Xylans are polysaccharides formed from 1 ,4-P-glycoside-linked D-xylopyranoses.

"Amylase" is polypeptide or peptide having multiple enzyme activities including the ability to hydrolyze internal alpha- 1 ,4-glucosidic linkages in starch to produce smaller molecular weight malto-dextrins. In one aspect, the a-amylase activity includes hydro lyzing internal alpha- 1,4-glucosidic linkages in starch at random. In another embodiment, the Amylase is a glucoamylase activity, a 1 ,4-a-D-glucan glucohydrolase activity, an exoamylase activity, or a β- amylase activity. The amylase activity can comprise hydrolyzing glucosidic bonds. In one aspect, the glucosidic bonds comprise an a- 1,4-glucosidic bond. In another aspect, the glucosidic bonds comprise an a-l,6-glucosidic bond. In one aspect, the amylase activity comprises hydrolyzing glucosidic bonds in starch, e.g., liquefied starch. The amylase activity can further comprise hydrolyzing glucosidic bonds into maltodextrins. In one aspect, the amylase activity comprises cleaving a maltose or a D-glucose unit from non-reducing end of the starch.

Pemulen 1622 polymer is a high molecular weight, crosslinked copolymer of acrylic acid and C10-C30 alkyl acrylate that is designed to form stable oil-in-water emulsions. It has relatively low viscosity compared to other Carbopol polymers

Pemulen 1621 polymer is a high molecular weight, crosslinked copolymer of acrylic acid and C10-C30 alkyl acrylate that is designed to form stable oil-in-water emulsions. It has relatively medium viscosity compared to other Pemulen polymers.

Carbopol ETD 2623 polymer is a hydrophobically modified, crosslinked polyacrylate powder.

Aculin 22 is an anionic hydrophobically-modified Alkali Soluble Emulsion (HASE). Acrylic acid (IUPAC: prop-2-enoic acid) is an organic compound with the formula CH2=CHC02H. It is the simplest unsaturated carboxylic acid, consisting of a vinyl group connected directly to a carboxylic acid terminus. Acrylic acid is also known as 2-Propenoic acid; Acroleic acid; glacial; Ethylenecarboxylic acid; Propenoic acid; Vinylformic acid. Acrylates/C10-C30 Alkyl acrylate crosspolymer

In one embodiment, a reversible enzyme inhibitor is a polymer with an inhibition constant (Kj) below 1 ug/mL range, wherein the enzyme is selected from a group consisting of: a phytase, a cellulase, a xylanase, and an amylase.

In another embodiment, the reversible enzyme inhibitor is a polymer, wherein the polymer is a cross-linked co-polymer comprising an acrylic acid and a C10-C30 alkyl acrylate.

In another embodiment, a reversible cellulase inhibitor is a composition comprising an acrylic acid and a C10-C30 alkyl acrylate with an inhibition constant (Κ;) below 1 ug/mL range.

In another embodiment, a reversible phytase inhibitor is a composition comprising an acrylic acid and a C10-C30 alkyl acrylate with an inhibition constant (Kj) below 1 ug/mL range.

In another embodiment, a formulation comprising: (a) a cellulase or a phytase; (b) an acrylic acid; and (c) a C10-C30 alkyl acrylate.

In another embodiment, a formulation is used in a gas or oil well fluid. In another embodiment, the formulation is used as an additive for an animal feed. In another embodiment, the formulation is used in a detergent. In another embodiment, the formulation is used in baking. In another embodiment, the formulation is used in the manufacturing of bio fuels.

All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention. All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. EXAMPLES Example 1 : Cellulase inhibition:

Cellulase I and Cellulase II at 20 ug/mL was mixed with Pemulenl622 (Lubrizol) at concentrations between 0.024-200,000 ng/mL (w/v) in 50 mM Na-acetate pH 5.0 and incubated at room temperature for 15 min. Enzyme control did not contain Pemulenl622 (Lubrizol). Samples were diluted 20 fold into 1% beta-glucan (Megazyme) plus 50 mM Na-acetate pH 5.0 containing no Pemulenl622 (Lubrizol) or containing the same amounts of Pemulenl622 (Lubrizol) as the stock solution. Activity was determined by DNS assay at 80°C, by stopping the reaction every 2 min, heating the plate at 80°C, followed by absorbance reading at 540 nm.

Pemulenl622 (Lubrizol) inhibited activities of Cellulase I and Cellulase II at concentrations above 0.5 ug/mL (Figure 1), without interfering with the spectroscopic properties of the detection assay (Figure 2).

Cellulase I recovered the enzymatic activity upon inhibitor dilution (Figure 3). These results, suggest that the mechanism of Cellulase inhibition was reversible.

Pemulen 1621 (Lubrizol), structurally similar to Pemulen 1622 (Lubrizol), was also shown to be a potent inhibitor of Cellulase I (Figure 4).

Carbopol ETD 2623 and Aculin 22, which are both structurally similar to Pemulein 1621 and Pemulen 1622 were also shown to be inhibitors of Cellulase I; however, these inhibitors were lower potency inhibitors of Cellulase I when compared to the inhibition potency of Pemulen 1622 and Pemulen 1621 (Figure 5).

Negative control enzymes - Xylanase I, Xylanase II, and Amylase I were used in similar conditions together with proper substrates 2% wheat arabinoxylan (Megazyme) and, respectively, 1% corn starch (Sigma). Glucose, mannose, and xylose standard curves were generated in presence and absence of Pemulenl622 (Lubrizol).

Pemulenl622 (Lubrizol) did not inhibit activity of Amylase I in the specified range. On the other hand, Pemulen 1622 (Lubrizol) produced mild inhibition for Xylanase I and Xylanase II at concentrations above 500 ug/mL (Figure 1). Example 2: Cellulase inhibition as a function of pH in PNP assay

Cellulase I (lU/mL) was incubated with Pemulen 1622 at the specified concentrations and pH 5-9 (acetate, citrate, Hepes, Tris, Bis-Tris). Samples were incubated at RT for 15 min, then diluted 10 fold into 2 mM PNP-glucopyranoside with same Pemulen 1622 (Lubrizol)

concentration and pH. Activity was measured at 65°C, reading absorbance at 405 nm.

Percentage of maximum activity (positive control= enzyme with no inhibitor) was plotted versus Pemulen 1622 concentration.

The results, shown in Figure 6, indicate that Pemulen 1622 (Lubrizol) inhibits Cellulase activity with high potency below about pH6 and with low potency above about pH7. Example 3: Phytase inhibition:

Phytase I at 6 ug/mL was mixed with Pemulen 1622 (Lubrizol) at concentrations between 0.024-200,000 ng/niL in 50 mM Na-acetate pH 5.0 and incubated at room temperature for 15 min. Enzyme control did not contain Pemulenl622. Samples were diluted 20 fold into 4 mM Phytic acid (Sigma) plus 50 mM Na-acetate pH 4.5, containing no Pemulen 1622 or containing the same amounts of Pemulenl622 (Lubrizol) as the stock solution. Activity was determined by phosphate colorimetric assay at 50°C, by stopping the reaction every 2 min and reading the absorbance at 415 nm. Phosphate standard curve was generated in presence and absence of Pemulenl622 (Lubrizol).

Pemulenl622 (Lubrizol) inhibited activity of Phytase I at concentrations above 0.5 ug/mL (Figure 1), without interfering with the spectroscopic properties of the detection assay (Figure 2).

Phytase I recovered the enzymatic activity upon inhibitor dilution (Figure 3). These results, suggest that the mechanism of Phytase inhibition was reversible.

Claims

CLAIMS What is claimed is:

1. A reversible enzyme inhibitor comprising: a polymer with an inhibition constant (Κ;) below 1 ug/mL, wherein the enzyme is a phytase or a cellulase.

2. The reversible enzyme inhibitor of claim 1 , wherein the polymer is a cross-linked co-polymer comprising an acrylic acid and a C10-C30 alkyl acrylate.

3. A reversible cellulase inhibitor comprising an acrylic acid and a C10-C30 alkyl acrylate, wherein the inhibitor has an inhibition constant (Ki) below 1 ug/mL.

4. A reversible phytase inhibitor comprising an acrylic acid and a C10-C30 alkyl acrylate, wherein the inhibitor has an inhibition constant (Ki) below 1 ug/mL.

5. A formulation comprising:

(a) a cellulase or a phytase;

(b) an acrylic acid; and

(c) a C10-C30 alkyl acrylate.

6. The formulation of claim 5, wherein the formulation is used is an industrial process comprising: a gas or oil well fluid; an animal feed additive; a detergent, a baking ingredient, or a bio fuel manufacturing process.