WO2022224279A1 - Archaeal neopullulanase variant and increased ph/thermo stability - Google Patents

Archaeal neopullulanase variant and increased ph/thermo stability Download PDF

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WO2022224279A1
WO2022224279A1 PCT/IR2021/050010 IR2021050010W WO2022224279A1 WO 2022224279 A1 WO2022224279 A1 WO 2022224279A1 IR 2021050010 W IR2021050010 W IR 2021050010W WO 2022224279 A1 WO2022224279 A1 WO 2022224279A1
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variant
neopullulanase
starch
fermentation product
containing material
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PCT/IR2021/050010
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French (fr)
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Seyedeh Farzaneh SEYEDEH FARZANEH
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Seyedeh Farzaneh Seyedeh Farzaneh
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Priority to PCT/IR2021/050010 priority Critical patent/WO2022224279A1/en
Publication of WO2022224279A1 publication Critical patent/WO2022224279A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01135—Neopullulanase (3.2.1.135)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present disclosure generally relates to biotechnology, genetic engineering, industrial enzyme production with application in pharmaceutical and food industry and particularly, production of cyclodextrin which has a
  • the present invention relates to variant neopullulanase derived from a parental novel archaeal thermostable neopullulanase originated from Desulfurococcus mucosus DSM 2162 cloned in E. coli BL21 (DE3) which is disclosed herein as SEQ ID NO: 1 .
  • the variant is disclosed herein as SEQ ID NO: 2.
  • the variant has improved properties compared to the parent. The improved properties are increased thermo stability pH stability.
  • the present invention relates to neopullulanase variant comprising a substitution at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1. wherein the variant has neopullulanase activity and wherein the variant has at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 and, wherein the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 80% relative activity at 70 °C, more particularly, at least 75% relative activity at 80 °C , more particularly at least 70% relative activity at 90 °C when measured at 75° C and, Wherein the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ⁇ 5, more particularly, at least 100% relative activity at pH ⁇ 6, 7
  • the present invention further relates to isolated poly nucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides. Also described is the use of neopullulanase of the invention for starch conversion to produce fermentation products.
  • the present invention also relates to uses of the variant of the invention in the pharmaceutical industry wide range of application in complexion materials in foods, pharmaceuticals, plastics, and agricultural products as emulsifiers, antioxidants, and stabilizing agents. More particularly, the polypeptide of the present invention may be further used for starch processes, in particular starch conversion, especially starch saccharification.
  • Hydrolyses are often considered as one of the most applicable enzymes in industry.
  • GHs glycoside hydrolases classified within the carbohydrate-Active enzyme (CAZy) database
  • CAZy carbohydrate-Active enzyme
  • the a-amylase family GH13 containing ⁇ 30 different enzyme specificities and more than 37000 sequences represents one of the largest GH families.
  • neopullulanase subfamilies (EC 3.2.1.42) was described as a group of enzymes which is a starch debranching enzyme having pullulan 6-glucano-hydrolase activity (EC3.2.1.41 ) that catalyzes hydrolyses the a-1 -6-glycosidic bonds in pullulan, releasing maltotriose with reducing carbohydrate ends.
  • Starch consists of almost 80% amylopectin and 20% amylose.
  • Amylopectin is a branched polysaccharide in which linear chains alpha-1 -4 D-glucose residues are joined by alpha-1 -6 glycosidic bonds
  • alpha-amylase partially hydrolyses amylopectin to produce branched and linear oligosaccharides. Prolonged degradation of amylopectin results in the formation of alpha-limit dextrin’s those are not susceptible to further degradation by the alpha-amylase.
  • Branched oligosaccharides can be hydrolyzed into linear oligosaccharides by a debranching enzyme like isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41 ).
  • pullulanase family can be classified into five different categories: (1) Type I, true pullulanases (EC 3.2.1.41), which hydrolyze a(1-6) glycosidic bonds producing maltotriose; (2) Type II, amylopullulanases (EC3.2.1 .41), which catalyze the activity of both a(1 -6) on pullulan and a(1 -4) glycosidic bonds on starch and produce maltotriose and mixture of glucose, maltose, and maltotriose, respectively; (3) pullulan hydrolase Type I, neopullulanase (EC 3.2.1.135), which can hydrolyze a(1 -4) and a(1-6) glycosidic linkages with high activity on pullulan and cyclodextrins, producing panose; (4) pullulan hydrolase II, isopullulanase (EC 3.2.1.57), with a(1 -
  • Neopullulanase has various industrial applications including but not limited to Saccharification of Starch, Production of High-Maltose Corn Syrup, Production of High-Fructose Corn Syrup, Starch Processing Industry, Detergent and Production of Cyclodextrins (CDs).
  • the present invention relates to variant neopullulanase derived from a parental novel archaeal thermostable neopullulanase originated from Desulfurococcus mucosus DSM 2162 cloned in E. coli BL21 (DE3) which is disclosed herein as SEQ ID NO: 1.
  • the variant is disclosed herein as SEQ ID NO: 2.
  • the variant has improved properties compared to the parent. The improved properties are increased thermo stability pH stability.
  • the present invention relates to neopullulanase variant comprising a substitution at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1.
  • the variant has neopullulanase activity and wherein the variant has at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 and, wherein the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 80% relative activity at 70 °C, more particularly, at least 75% relative activity at 80 °C , more particularly at least 70% relative activity at 90 °C when measured at 75° C and, Wherein the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ⁇ 5, more particularly, at least 100% relative activity at pH ⁇ 6, 7 and 8, more particularly, at least 75% relative activity at pH ⁇ 9, more particularly, at least 70% relative activity at pH ⁇ 10 when comparing parent of SEQ ID NO: 1 .
  • the present invention further relates to isolated poly nucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides. Also described is the use of neopullulanase of the invention for starch conversion to produce fermentation products. The present invention also relates to uses of the variant of the invention in the pharmaceutical industry.
  • the present invention provides neopullulanase variant with improved properties compared to its parent. It is an object of the present invention to provide novel neopullulanase variant having an increased stability at low pH and / or at high temperature.
  • amino acid 33 of neopullulanase variant substitude wherein histidine substitute with glycine at the polypeptide sequence ID NO.:1 to obtain the polypeptide sequence ID No.: 2.
  • the variant has neopullulanase activity; and wherein the variant has at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 and wherein the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 70% relative activity at 60 °C, at least 60% relative activity at 70 °C , at least 50% relative activity at 80 °C when measured at 75° C and wherein the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ⁇ 5, at least 100% relative activity at pH ⁇ 6, 7 and 8, at least 75% relative activity at pH ⁇ 9, at least 70% relative activity at pH ⁇ 10.
  • the variant has improved properties compared to the parent.
  • the improved properties are increased thermo stability and pH stability neopullulanase variant of the present invention, comprising a substitution at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1 . wherein the variant has neopullulanase activity and wherein the variant has at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 , and
  • the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 70% relative activity at 60 °C, more particularly, at least 60% relative activity at 70 °C , more particularly at least 50% relative activity at 80 °C when measured at 75° C and,
  • the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ⁇ 5, more particularly, at least 100% relative activity at pH ⁇ 6, 7 and 8, more particularly, at least 75% relative activity at pH ⁇ 9, more particularly, at least 70% relative activity at pH ⁇ 10 when comparing parent of SEQ ID NO: 1 .
  • FIG. 1 A A first figure.
  • FIG.1 A illustrates the polynucleotide sequence ID NO. 1 which is related to the parent neopullulanase.
  • FIG.1 B illustrates the polynucleotide sequence ID NO. 2 which is related to the neopullulanase variant of the present invention.
  • FIG.2A illustrates the polypeptide sequence ID NO. 1 which is related to the parent neopullulanase
  • FIG. 2B [0019] [fig.2B] illustrates the polypeptide sequence ID NO. 2 which is related to the neopullulanase variant of the present invention.
  • FIG.3 illustrates the nucleic acid construct comprising the polynucleotide sequence of the present invention.
  • FIG.4 illustrates the substrate preference of the neopullulanase variant of the present invention.
  • FIG.5A illustrates the thermo-stability of the parent neopullulanase.
  • FIG.5B illustrates the increased thermo-stability of the neopullulanase variant of the present invention.
  • FIG.6A illustrates the pH stability of the parent neopullulanase.
  • FIG.6B illustrates the increased pH of the neopullulanase variant of the present invention.
  • a neopullulanase variant comprising a substitution at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1 wherein the variant has pullulanase activity;
  • the variant has at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 .
  • the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 70% relative activity at 60 °C, more particularly, at least 60% relative activity at 70 °C , more particularly at least 50% relative activity at 80 °C when measured at 75° C and,
  • the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ⁇ 5, more particularly, at least 100% relative activity at pH ⁇ 6, 7 and 8, more particularly, at least 75% relative activity at pH ⁇ 9, more particularly, at least 70% relative activity at pH ⁇ 10 when comparing p of SEQ ID NO: 1.
  • Embodiment 2 A method for producing a variant neopullulanase of a parent neopullulanase comprising substitution of the parent neopullulanase at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1 wherein the variant has pullulanase activity; and wherein the variant has at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
  • Embodiment 3 A use of a variant neopullulanase of embodiments 1 for production of a syrup and/or a fermentation product, e.g., ethanol, from a starch containing material.
  • a fermentation product e.g., ethanol
  • step (a) and/or step (b) Fermenting with a fermenting organism; wherein step (a) and/or step (b) is carried out in the presence of a variant neopullulanase of any of embodiments 1.
  • starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material
  • step (a) is carried out using at least a glucoamylase, and a variant neopullulanase of any of embodiments 1 .
  • Embodiment 12 An expression vector comprising the polynucleotide of embodiment 10.
  • Embodiment 14 A method of producing a neopullulanase variant of embodiment 1 , comprising cultivating the host cell of embodiment 13 under conditions conducive for production of the polypeptide.
  • M M (Molar); mol (moles); mmol (millimoles); prrnol (micromoles); pmol (picomoles); g (grams); mg (milligrams); ng (nanograms); L (liters); ml (milliliters); mI (microliters); M (molar); mM (millimolar); mM (micromolar); U (unit).
  • the purified genomic DNA of Desulfurococcus mucosus DSM 2162 was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ).
  • the neopullulanase gene fragment was amplified by PCR technique using primers containing Ndel and Hindi 11 restriction sites.
  • AMDMF 5'-ATCGCATATGGAAAGAATAATAGGGTATCTCGAG-3'
  • AMDMR 5'-ATCGAAGCTTAAGGGCTCCACCACCCCTGTAG-3'
  • the resultant PCR fragments were purified by NucleoSpin® Gel and PCR Clean-up kit [MACFIEREY-NAGEL).
  • the PCR product fragment and pET-22b(+) vector (Novagen, Darmstadt, Germany) were digested by Ndel and Hindlll restriction enzymes, then electrophoresed and extracted from 1% agarose gel by Silica Bead DNA Gel Extraction Kit - (Thermo Fisher Scientific) .
  • ligation of PCR products to Ndel/Hindl I l-digested pET-22b (+) utilizing T4 DNA ligase at 16 °C overnight, transformation into the cloning host, E. coli XL1-Blue cells.
  • Host cells comprising the recombinant plasmids were grown in LB agar plates supplemented by ampicillin (100 pg ml 1).
  • the recombinant constructed neopullulanase vector was extracted from host cells by Biospin plasmid DNA extraction kit (Bioflux, China) and tentatively named as DSMA. Correct insertion was analyzed by 1% agarose gel electrophoresis of double digested recombinant plasmid and further confirmed by DNA sequencing (Macrogen, South Korea).
  • a reverse or forward primer having desired mutation at target site (indicated with bold characters) were designed and Quick-change PCR was carried out using Primed FWQ and RVQ using DSMA vector as template.
  • RVQ GT CT CCACT ACCCCCCAG AAGCT CT AG
  • reaction mixture was divided into two and 10 pmol of each primer was added to each one of the mixtures.
  • D. mucosus DSM 2162 neopullulanase was performed in E. coli BL21 (DE3) comprising the recombinant constructed vector (pET-22b(+)-DSMA).
  • the recombinant constructed vector pET-22b(+)-DSMA.
  • E. coli cells were grown aerobically at 37 °C and under 200 rpm shaking condition in a pre-culture medium overnight.
  • Host cells comprising DSMA vector and its variant were inoculated (1%) into production medium and further cultured at 37 °C for 24 h at 250 rpm.
  • the host cells comprising DSMA vector and its variant were harvested by centrifugation (8500 xg for 5 min) and resuspended in 1/20 volume of lysis buffer
  • the host cells comprising DMSA vector and its variant disruption was conducted by sonication (60 kW) for 15 min in 30 S intervals for a 20 ml sample (Bandelin electronic GmbH & Co); the cell debris was removed by centrifugation at 8500 xg, 10 min, 4 °C (Sigma 2-16PK), and the cell free supernatant was kept at 4 °C for further analysis.
  • Neopullulanase and its variant were heated at 70 °C for 15 minutes, cooled on ice for 30 minutes, centrifuged at 8500 xg at 4 °C for 10 minutes.
  • the enzymatic crud mixture was loaded onto Ni-NTA agarose (Qiagen) affinity column chromatography (20 x 10 mm) and eluted with various concentrations of imidazole (5, 25, 75, 150, 200 and 250 mM) in the lysis buffer, pH ⁇ 5.5.
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was finally performed to analyze fractions according to the standard protocols known in the art.
  • Neopullulanase and its variant fraction were obtained when the lysis buffer with 250 mM of imidazole was used. Neopullulanase and its variant were further dialyzed against sodium acetate 50 mM; NaCI 50 mM, pH ⁇ 5.5 with the buffer being replaced three times for 24 hours. The protein concentration was determined by Bradford assay using BSA as standard in accordance with the methods well known in the art.
  • reaction mixture was prepared in accordance with the following:
  • the colored sample was diluted by cold distilled water 10 times and the absorbance was recorded at 540 nm.
  • Neopullulanase activity was defined as the amount of enzyme required to produce 1 mole of D-maltose under standard assay conditions.
  • Substrate specificity of neopullulanase and its variant was evaluated using HPLC technique using AZURA column (KNAUER, Germany). The HPLC grade water was applied as the mobile phase with 0.5 ml/min flow rate according to methods known in the art. The neopullulanase was incubated at optimum temperatures with 0.5% (W/V) pullulan, starch, b-cyclodextrin, or amylose. The enzymatic reaction of all samples was stopped by incubation on iced cold water. Products of enzymatic reaction were analyzed using appropriate standards.
  • Neopullulanase activities were measured at several temperatures ranging from 40 to 90°C by soluble starch assay described in EXAMPLE 4. Relative activity of neopullulanase variants showing higher thermal activity compared to parental neopullulanase. Relative activity is Relative activity is the ratio between the activity of samples of interest and the activity of the control sample and therefore expressed as a percentage.
  • neopullulanase activities were measured at several pH values ranging from 3.5 to 10.0, in a mixed buffer containing sodium acetate 25 mM, sodium phosphate 25 mM, Tris-base 25 mM, and glycine-NaOH 25 mM) at a constant temperature of 70°C.by soluble starch assay described in EXAMPLE 4. Relative activity measured as described in EXAMPLE 6.
  • Neopullulanase and its variants were pre-incubated in sodium acetate buffer 50 mM, pH ⁇ 5.5 at 60, 70, and 80 °C for 1 hour in the presence and absence of 5 mM Ca+2 cation followed by cooling the enzyme mixture at 4 °C for 30 minutes.
  • Neopullulanase activities were measured by soluble starch assay described in EXAMPLE 4.
  • the enzymatic activity without pre-incubation was regarded as control (100% remaining activity). Relative activity measured as described in EXAMPLE 6.
  • Neopullulanase and its variants were pre-incubated at room temperature for 1 h in the mix buffer with pH values ranging from 4 to 10.
  • Neopullulanase activities were measured by soluble starch assay described in EXAMPLE 4.
  • the enzymatic activity without pre-incubation was regarded as control (100% remaining activity). Relative activity measured as described in EXAMPLE 6.
  • Neopullulanase and its variant were pre-incubated the mentioned additives at 30 °C for 30 minutes; 1 and 5 mM of Ca2+, Mg2+, Mn2+, Fe3+, Co2+, Cu2+, Zn2+, Ni2+, or Hg2+ and EDTA, Triton X-100 at 5 and 10 mM, and SDS 1%.
  • Neopullulanase activities were measured by soluble starch assay described in EXAMPLE 4. The enzymatic activity without any additive was regarded as control. Relative activity measured as described in example 6.
  • the variants may be used in production of cyclodextrin in which has a wide range of application in complexion materials in foods, pharmaceuticals, plastics, and agricultural products as emulsifiers, antioxidants, and stabilizing agents.
  • variants may be used in starch processing applications especially, starch saccharification and Liquefaction.
  • the present invention also can be used in the production of sweeteners and production ethanol, such as fuel, drinking and industrial ethanol, from starch or whole grains.
  • the inventors have used methods in the art of genetic engineering in particular site direct mutagenesis to alter the amino acid at position 33 at the sequence ID No. 1 to obtain the sequence ID No. 2, wherein histidin is substitute with glycine, where in the variant has neopullulanase activity; and the variant has at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
  • neopullulanse variant of the present invention was determined as described here in at example 5. Hydrolytic activity was monitored using five different substrates: starch, pullulan, b-cyclodextrin, amylopectin, and glycogen. The optimum enzymatic activity was seen on the pullulan substrate, followed by b-cyclodextrin (68%) and amylopectin (51%). The hydrolytic activity on starch and glycogen was less than 30% of its maximum activity on pullulan.
  • the increased productivity of neopullulanase variant was evaluated as described herein at examples of 6-10.
  • the neopullulanase variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 70% relative activity at 60 °C, at least 60% relative activity at 70 °C , at least 50% relative activity at 80 °C when measured at 75° C .
  • the neopullulanase variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ⁇ 5, at least 100% relative activity at pH ⁇ 6, 7 and 8, at least 75% relative activity at pH ⁇ 9, at least 70% relative activity at pH ⁇ 10.
  • NPL1 Hue, K. K procedura Cohen, S. D Struktur & Bechhofer, D. H. (1995). A polypurine sequence that acts as a 5'mRNA stabilizer in Bacillus subtilis. Journal of bacteriology, 777(12), 3465-3471.
  • NPL2 Simonen, M., & Palva, I. (1993). Protein secretion in Bacillus species. Microbiology and Molecular Biology Reviews, 57(1 ), 109-137.

Abstract

The invention relates to novel variant of the enzymatic peptide neopullulanase, the gene sequences encoding said novel peptides, expression vectors comprising those gene sequences as well as organisms expressing the novel neopullulanase variant. The novel neopullulanase variant of the present invention was made empirically by the use site direct mutagenesis and substitution in one position corresponding to position (33) of "wild-type" polypeptide. Furthermore, the invention relates to the use of these novel neopullulanase peptides in the pharmaceutical, textile, fermentation, food, and other industries.

Description

WO 2022/224279 DeSCNptiOll PCT/IR2021/050010
Title of Invention :
ARCHAEAL NEOPULLULANASE VARIANT AND INCREASED PH/THERMO STABILITY
Technical Field
[0002] The present disclosure generally relates to biotechnology, genetic engineering, industrial enzyme production with application in pharmaceutical and food industry and particularly, production of cyclodextrin which has a The present invention relates to variant neopullulanase derived from a parental novel archaeal thermostable neopullulanase originated from Desulfurococcus mucosus DSM 2162 cloned in E. coli BL21 (DE3) which is disclosed herein as SEQ ID NO: 1 . The variant is disclosed herein as SEQ ID NO: 2. In one aspect, the variant has improved properties compared to the parent. The improved properties are increased thermo stability pH stability. In another aspect, the present invention relates to neopullulanase variant comprising a substitution at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1. wherein the variant has neopullulanase activity and wherein the variant has at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 and, Wherein the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 80% relative activity at 70 °C, more particularly, at least 75% relative activity at 80 °C , more particularly at least 70% relative activity at 90 °C when measured at 75° C and, Wherein the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ~5, more particularly, at least 100% relative activity at pH ~6, 7 and 8, more particularly, at least 75% relative activity at pH ~9, more particularly, at least 70% relative activity at pH ~10 when comparing parent of SEQ ID NO: 1 . In another aspect, the present invention further relates to isolated poly nucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides. Also described is the use of neopullulanase of the invention for starch conversion to produce fermentation products. The present invention also relates to uses of the variant of the invention in the pharmaceutical industry wide range of application in complexion materials in foods, pharmaceuticals, plastics, and agricultural products as emulsifiers, antioxidants, and stabilizing agents. More particularly, the polypeptide of the present invention may be further used for starch processes, in particular starch conversion, especially starch saccharification.
Background Art
[0003] Hydrolyses are often considered as one of the most applicable enzymes in industry. Among the glycoside hydrolases (GHs) classified within the carbohydrate-Active enzyme (CAZy) database, the a-amylase family GH13 containing ~30 different enzyme specificities and more than 37000 sequences represents one of the largest GH families. Based on a characteristic sequence motif in their fifth conserved sequence region the neopullulanase subfamilies (EC 3.2.1.42) was described as a group of enzymes which is a starch debranching enzyme having pullulan 6-glucano-hydrolase activity (EC3.2.1.41 ) that catalyzes hydrolyses the a-1 -6-glycosidic bonds in pullulan, releasing maltotriose with reducing carbohydrate ends.
[0004] Starch consists of almost 80% amylopectin and 20% amylose. Amylopectin is a branched polysaccharide in which linear chains alpha-1 -4 D-glucose residues are joined by alpha-1 -6 glycosidic bonds alpha-amylase partially hydrolyses amylopectin to produce branched and linear oligosaccharides. Prolonged degradation of amylopectin results in the formation of alpha-limit dextrin’s those are not susceptible to further degradation by the alpha-amylase. Branched oligosaccharides can be hydrolyzed into linear oligosaccharides by a debranching enzyme like isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41 ).
[0005] Based on the substrate specificity and product diversity, pullulanase family can be classified into five different categories: (1) Type I, true pullulanases (EC 3.2.1.41), which hydrolyze a(1-6) glycosidic bonds producing maltotriose; (2) Type II, amylopullulanases (EC3.2.1 .41), which catalyze the activity of both a(1 -6) on pullulan and a(1 -4) glycosidic bonds on starch and produce maltotriose and mixture of glucose, maltose, and maltotriose, respectively; (3) pullulan hydrolase Type I, neopullulanase (EC 3.2.1.135), which can hydrolyze a(1 -4) and a(1-6) glycosidic linkages with high activity on pullulan and cyclodextrins, producing panose; (4) pullulan hydrolase II, isopullulanase (EC 3.2.1.57), with a(1 -4) glycosidic bond hydrolyzing activity on cyclodextrins with the end product of isopanose, and (5) pullulan hydrolase III with a broad spectrum activity to degrade both a(1 -4) and a(1-6) glycosidic linkages to form a mixture of panose, glucose, maltose, and maltotriose.
[0006] Neopullulanase has various industrial applications including but not limited to Saccharification of Starch, Production of High-Maltose Corn Syrup, Production of High-Fructose Corn Syrup, Starch Processing Industry, Detergent and Production of Cyclodextrins (CDs).
[0007] [0006] Pullulanases are known in the art. U.S. Pat. Nos. 6,074,854 and 5,817,498 disclose a pullulanase from Bacillus deramificans. W02009/075682 discloses a pullulanase derived from Bacillus acidopullulyticus. W095/23852 discloses an amylopululanase from Thermococcus caller and the use for producing 5 Sweeteners and ethanol from starch. US 8,354,101 B2 disclose pullulanase with improved productivity. WO2015110473A3 Discloses pullulanase variants and polynucleotides encoding same. US 8,703,465 B2 disclose pullulanase variants and uses.
Summary of Invention
[0008] The present invention relates to variant neopullulanase derived from a parental novel archaeal thermostable neopullulanase originated from Desulfurococcus mucosus DSM 2162 cloned in E. coli BL21 (DE3) which is disclosed herein as SEQ ID NO: 1. The variant is disclosed herein as SEQ ID NO: 2. In one aspect, the variant has improved properties compared to the parent. The improved properties are increased thermo stability pH stability. In another aspect, the present invention relates to neopullulanase variant comprising a substitution at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1. wherein the variant has neopullulanase activity and wherein the variant has at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 and, Wherein the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 80% relative activity at 70 °C, more particularly, at least 75% relative activity at 80 °C , more particularly at least 70% relative activity at 90 °C when measured at 75° C and, Wherein the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ~5, more particularly, at least 100% relative activity at pH ~6, 7 and 8, more particularly, at least 75% relative activity at pH ~9, more particularly, at least 70% relative activity at pH ~10 when comparing parent of SEQ ID NO: 1 . In another aspect, the present invention further relates to isolated poly nucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides. Also described is the use of neopullulanase of the invention for starch conversion to produce fermentation products. The present invention also relates to uses of the variant of the invention in the pharmaceutical industry.
Technical Problem
[0009] Thermo stability and pH stability is of great importance when comes to enzymes with industrial application. Such enzymes required improved properties using methods in the art of genetic engineering.
Solution to Problem
[0010] The present invention provides neopullulanase variant with improved properties compared to its parent. It is an object of the present invention to provide novel neopullulanase variant having an increased stability at low pH and / or at high temperature.
[0011 ] In the present invention, the amino acid 33 of neopullulanase variant substitude, wherein histidine substitute with glycine at the polypeptide sequence ID NO.:1 to obtain the polypeptide sequence ID No.: 2. Wherein the variant has neopullulanase activity; and wherein the variant has at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 and wherein the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 70% relative activity at 60 °C, at least 60% relative activity at 70 °C , at least 50% relative activity at 80 °C when measured at 75° C and wherein the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ~5, at least 100% relative activity at pH ~6, 7 and 8, at least 75% relative activity at pH ~9, at least 70% relative activity at pH ~10.
Advantageous Effects of Invention [0012] In accordance with the present invention the variant has improved properties compared to the parent. The improved properties are increased thermo stability and pH stability neopullulanase variant of the present invention, comprising a substitution at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1 . wherein the variant has neopullulanase activity and wherein the variant has at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 , and
[0013] a) Wherein the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 70% relative activity at 60 °C, more particularly, at least 60% relative activity at 70 °C , more particularly at least 50% relative activity at 80 °C when measured at 75° C and,
[0014] b) Wherein the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ~5, more particularly, at least 100% relative activity at pH ~6, 7 and 8, more particularly, at least 75% relative activity at pH ~9, more particularly, at least 70% relative activity at pH ~10 when comparing parent of SEQ ID NO: 1 .
Brief Description of Drawings
[0015] The following drawings are illustrative of embodiments of the invention and are not meant to limit the Scope of the invention as encompassed by the claims.
FIG. 1 A
[0016] [fig.1 A] illustrates the polynucleotide sequence ID NO. 1 which is related to the parent neopullulanase.
FIG. 1 B
[0017] [fig.1 B] illustrates the polynucleotide sequence ID NO. 2 which is related to the neopullulanase variant of the present invention.
FIG. 2A
[0018] [fig.2A] illustrates the polypeptide sequence ID NO. 1 which is related to the parent neopullulanase
FIG. 2B [0019] [fig.2B] illustrates the polypeptide sequence ID NO. 2 which is related to the neopullulanase variant of the present invention.
FIG. 3
[0020] [fig.3] illustrates the nucleic acid construct comprising the polynucleotide sequence of the present invention.
FIG. 4
[0021] [fig.4] illustrates the substrate preference of the neopullulanase variant of the present invention.
FIG. 5A
[0022] [fig.5A] illustrates the thermo-stability of the parent neopullulanase.
FIG. 5B
[0023] [fig.5B] illustrates the increased thermo-stability of the neopullulanase variant of the present invention.
FIG. 6A
[0024] [fig.6A] illustrates the pH stability of the parent neopullulanase.
FIG. 6B
[0025] [fig.6B] illustrates the increased pH of the neopullulanase variant of the present invention.
Description of Embodiments
[0026] The present invention is further described by the following numbered embodiments:
[0027] A neopullulanase variant comprising a substitution at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1 wherein the variant has pullulanase activity; and
[0028] Wherein the variant has at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 , and
[0029] Wherein the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 70% relative activity at 60 °C, more particularly, at least 60% relative activity at 70 °C , more particularly at least 50% relative activity at 80 °C when measured at 75° C and,
[0030] Wherein the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ~5, more particularly, at least 100% relative activity at pH ~6, 7 and 8, more particularly, at least 75% relative activity at pH ~9, more particularly, at least 70% relative activity at pH ~10 when comparing p of SEQ ID NO: 1.
[0031] [Embodiment 2] A method for producing a variant neopullulanase of a parent neopullulanase comprising substitution of the parent neopullulanase at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1 wherein the variant has pullulanase activity; and wherein the variant has at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
[0032] [Embodiment 3] A use of a variant neopullulanase of embodiments 1 for production of a syrup and/or a fermentation product, e.g., ethanol, from a starch containing material.
[0033] [Embodiment 4] The use according to embodiment 1 , wherein the starch material is gelatinized or un-gelatinized starch material.
[0034] [Embodiment 5] A process of producing a fermentation product from starch- containing material comprising the steps of:
[0035] Liquefying starch-containing material in the presence of an alpha amylase.
[0036] Saccharifying the liquefied material in the presence of a glucoamylase; and
[0037] Fermenting with a fermenting organism; wherein step (a) and/or step (b) is carried out in the presence of a variant neopullulanase of any of embodiments 1.
[0038] [Embodiment 6] A process of producing a fermentation product from starch- containing material, comprising the steps of:
[0039] starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; and
[0040] Fermenting with a fermenting organism [0041] Wherein step (a) is carried out using at least a glucoamylase, and a variant neopullulanase of any of embodiments 1 .
[0042] [Embodiment 7] The process according to embodiment 6, wherein an alpha amylase is added in step (a).
[0043] [Embodiment 8] The process according to embodiment 6, wherein saccharification and fermentation is carried out simultaneously.
[0044] [Embodiment 9] The process according to any of embodiments 5-8, wherein the fermentation product is an alcohol, particularly ethanol.
[0045] [Embodiment 10] A polynucleotide encoding the variant neopullulanase of embodiments 1 .
[0046] [Embodiment 11] A nucleic acid construct comprising the polynucleotide of embodiment 10.
[0047] Embodiment 12] An expression vector comprising the polynucleotide of embodiment 10.
[0048] [Embodiment 13] A host cell comprising the polynucleotide of embodiment 10.
[0049] [Embodiment 14] A method of producing a neopullulanase variant of embodiment 1 , comprising cultivating the host cell of embodiment 13 under conditions conducive for production of the polypeptide.
[0050] [Embodiment 15] The method of embodiment 14, further comprising recovering the polypeptide.
[0051] [Embodiment 16] A whole broth formulation or cell culture composition comprising a polypeptide of any of embodiments 1 .
Examples
[0052] In the experimental disclosure which follows, the following abbreviations apply: M (Molar); mol (moles); mmol (millimoles); prrnol (micromoles); pmol (picomoles); g (grams); mg (milligrams); ng (nanograms); L (liters); ml (milliliters); mI (microliters); M (molar); mM (millimolar); mM (micromolar); U (unit).
[0053] The present invention is described in further details in the following examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein. The following examples are offered to illustrate, but not to limit the claimed invention.
[0054] Examples 1
[0055] Construction of Parent Neopullulanase
[0056] The purified genomic DNA of Desulfurococcus mucosus DSM 2162 was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ). The neopullulanase gene fragment was amplified by PCR technique using primers containing Ndel and Hindi 11 restriction sites.
[0057] AMDMF: 5'-ATCGCATATGGAAAGAATAATAGGGTATCTCGAG-3'
[0058] AMDMR: 5'-ATCGAAGCTTAAGGGCTCCACCACCCCTGTAG-3'
[0059] The PCR reaction mixture was prepared as follow:
[0060] PCR buffer 10mI
[0061] MgCI22.5 pL of 2.5 mM solution
[0062] Template Desulfurococcus mucosus DSM 2162 purified genomic DNA 10 ng
[0063] dNTP mixture 1 pL of 10mM solution
[0064] Taq DNA polymerase 1 Unit
[0065] Reach the final concentration of 50 pL with distilled water.
[0066] The PCR reaction was carried out as follow:
[0067] Initial denaturation 94 °C, 3 minutes
[0068] 35 cycle of: 94 °C 60 seconds, 56 °C 60 seconds, 72 °C 120 seconds
[0069] Final elongation 72 °C, 10 minutes
[0070] The resultant PCR fragments were purified by NucleoSpin® Gel and PCR Clean-up kit [MACFIEREY-NAGEL). The PCR product fragment and pET-22b(+) vector (Novagen, Darmstadt, Germany) were digested by Ndel and Hindlll restriction enzymes, then electrophoresed and extracted from 1% agarose gel by Silica Bead DNA Gel Extraction Kit - (Thermo Fisher Scientific) . Followed by ligation of PCR products to Ndel/Hindl I l-digested pET-22b (+) utilizing T4 DNA ligase at 16 °C overnight, transformation into the cloning host, E. coli XL1-Blue cells. Host cells comprising the recombinant plasmids were grown in LB agar plates supplemented by ampicillin (100 pg ml 1). The recombinant constructed neopullulanase vector was extracted from host cells by Biospin plasmid DNA extraction kit (Bioflux, China) and tentatively named as DSMA. Correct insertion was analyzed by 1% agarose gel electrophoresis of double digested recombinant plasmid and further confirmed by DNA sequencing (Macrogen, South Korea).
[0071] Example 2
[0072] Construction of Improved Variant of Neopullulanase.
[0073] A reverse or forward primer having desired mutation at target site (indicated with bold characters) were designed and Quick-change PCR was carried out using Primed FWQ and RVQ using DSMA vector as template.
[0074] FWQ: CT AG AGCTT CTGGGGGGT AGTGG AG AC
[0075] RVQ: GT CT CCACT ACCCCCCAG AAGCT CT AG
[0076] The PCR reaction mixture was prepared as follow:
[0077] PCR buffer containing MgCI2 15mI
[0078] Template DSMA plasmid 100 ng
[0079] dNTP mixture 6pL of 10mM solution
[0080] Prime STAR HS DNA polymerase 5 Unit
[0081] Reach the final concentration of 49 pL with distilled water
[0082] The reaction mixture was divided into two and 10 pmol of each primer was added to each one of the mixtures.
[0083] The PCR reaction was carried out as follow:
[0084] Starting denaturation 95 °C, 5 minutes
[0085] 6 cycle of: 95 °C 30 second, 60 °C 30 s, 68 °C 10 minutes
[0086] Two mixtures were mixed into one and the following was carried out:
[0087] 24 cycle of: 95 °C 30 seconds, 60 °C 30 seconds, 68 °C 10 minutes
[0088] Final elongation 68 °C, 10 minutes [0089] The resultant PCR fragments were purified by NucleoSpin® Gel and PCR Clean-up kit [MACHEREY-NAGEL], treated with DpN1 enzyme and introduced into E. coli.
[0090] Example 3
[0091] Neopullulanase and Its Variant Expression in E. coli BL21 (DE3)
[0092] The overexpression of D. mucosus DSM 2162 neopullulanase (DSMA) was performed in E. coli BL21 (DE3) comprising the recombinant constructed vector (pET-22b(+)-DSMA). The recombinant
[0093] E. coli cells were grown aerobically at 37 °C and under 200 rpm shaking condition in a pre-culture medium overnight.
[0094] Pre culture medium:
[0095] Glucose 5 g.L-1
[0096] KH2P0450 mM
[0097] Na2HP0450 mM
[0098] (NH4)2S0425 mM
[0099] MgS042 mM
[0100] Trace elements 5%
[0101] Ampicillin 100 mg ml 1
[0102] Host cells comprising DSMA vector and its variant were inoculated (1%) into production medium and further cultured at 37 °C for 24 h at 250 rpm.
[0103] Production medium ZYM5052:
[0104] Yeast extracts 5 g.L-1
[0105] Tryptone 10 g.L-1
[0106] Glucose 0.5 g. L-1
[0107] a-D-lactose 2 g.L-1
[0108] KH2P0425 mM
[0109] Na2HP0425 mM [0110] NH4CI 50 mM [0111] Na2S045 mM [0112] MgS042 mM [0113] Glycerol 0.5%
[0114] Trace elements 5%
[0115] Ampicillin 100 mg ml 1
[0116] the host cells comprising DSMA vector and its variant were harvested by centrifugation (8500 xg for 5 min) and resuspended in 1/20 volume of lysis buffer
[0117] Lysis Buffer:
[0118] Sodium acetate 50 mM
[0119] NaCI 50 mM
[0120] pH ~ 5.5
[0121] The host cells comprising DMSA vector and its variant disruption was conducted by sonication (60 kW) for 15 min in 30 S intervals for a 20 ml sample (Bandelin electronic GmbH & Co); the cell debris was removed by centrifugation at 8500 xg, 10 min, 4 °C (Sigma 2-16PK), and the cell free supernatant was kept at 4 °C for further analysis.
[01221 EXAMPLE 4
[0123] Neopullulanase and Its Variant Purification
[0124] The Cell free supernatant containing Neopullulanase and its variant were heated at 70 °C for 15 minutes, cooled on ice for 30 minutes, centrifuged at 8500 xg at 4 °C for 10 minutes. The enzymatic crud mixture was loaded onto Ni-NTA agarose (Qiagen) affinity column chromatography (20 x 10 mm) and eluted with various concentrations of imidazole (5, 25, 75, 150, 200 and 250 mM) in the lysis buffer, pH ~ 5.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was finally performed to analyze fractions according to the standard protocols known in the art. The highly purified Neopullulanase and its variant fraction were obtained when the lysis buffer with 250 mM of imidazole was used. Neopullulanase and its variant were further dialyzed against sodium acetate 50 mM; NaCI 50 mM, pH ~ 5.5 with the buffer being replaced three times for 24 hours. The protein concentration was determined by Bradford assay using BSA as standard in accordance with the methods well known in the art.
[0125]
[0126] Example 5
[0127] Neopullulanase and Its Variant Enzymatic Assay
[0128] The enzyme activity and substrate specificity were captured using the followings as substrate:
[0129] Soluble starch
[0130] Amylopectin
[0131] Glycogen
[0132] b-cyclodextrin
[0133] Pullulan
[0134] The reaction mixture was prepared in accordance with the following:
[0135] 20 mI of appropriately diluted Neopullulanase or its variant
[0136] 230 mI of 1% various substrates (pH ~ 5.5)
[0137] Reaction mixture was incubated for 15 minutes at 70 °C
[0138] The reducing activity of the end products was quantified by adding 250 mI of DNS reagent and boiling for 5 minutes.
[0139] The colored sample was diluted by cold distilled water 10 times and the absorbance was recorded at 540 nm.
[0140] Various concentrations of D-maltose were used to make a standard curve.
[0141] One unit of Neopullulanase activity was defined as the amount of enzyme required to produce 1 mole of D-maltose under standard assay conditions.
[0142] EXAMPLE 5
[0143] Substrate specificity of neopullulanase and its variant was evaluated using HPLC technique using AZURA column (KNAUER, Germany). The HPLC grade water was applied as the mobile phase with 0.5 ml/min flow rate according to methods known in the art. The neopullulanase was incubated at optimum temperatures with 0.5% (W/V) pullulan, starch, b-cyclodextrin, or amylose. The enzymatic reaction of all samples was stopped by incubation on iced cold water. Products of enzymatic reaction were analyzed using appropriate standards.
[0144] Example 6
[0145] Evaluation of Thermal activity of Neopullulanase
[0146] Neopullulanase activities were measured at several temperatures ranging from 40 to 90°C by soluble starch assay described in EXAMPLE 4. Relative activity of neopullulanase variants showing higher thermal activity compared to parental neopullulanase. Relative activity is Relative activity is the ratio between the activity of samples of interest and the activity of the control sample and therefore expressed as a percentage.
[0147] EXAMPLE 7
[0148] Evaluation of pH activity of Neopullulanase
[0149] neopullulanase activities were measured at several pH values ranging from 3.5 to 10.0, in a mixed buffer containing sodium acetate 25 mM, sodium phosphate 25 mM, Tris-base 25 mM, and glycine-NaOH 25 mM) at a constant temperature of 70°C.by soluble starch assay described in EXAMPLE 4. Relative activity measured as described in EXAMPLE 6.
[0150] Example 8
[0151] Evaluation of Thermo-stability of Neopullulanase
[0152] The neopullulanase and its variants were pre-incubated in sodium acetate buffer 50 mM, pH ~ 5.5 at 60, 70, and 80 °C for 1 hour in the presence and absence of 5 mM Ca+2 cation followed by cooling the enzyme mixture at 4 °C for 30 minutes. Neopullulanase activities were measured by soluble starch assay described in EXAMPLE 4. The enzymatic activity without pre-incubation was regarded as control (100% remaining activity). Relative activity measured as described in EXAMPLE 6.
[0153] EXAMPLE 9 [0154] Evaluation of pH stability of Neopullulanase
[0155] The neopullulanase and its variants were pre-incubated at room temperature for 1 h in the mix buffer with pH values ranging from 4 to 10. Neopullulanase activities were measured by soluble starch assay described in EXAMPLE 4. The enzymatic activity without pre-incubation was regarded as control (100% remaining activity). Relative activity measured as described in EXAMPLE 6.
[0156] Example 10
[0157] Evaluation of Additives on Neopullulanase Activity
[0158] Neopullulanase and its variant were pre-incubated the mentioned additives at 30 °C for 30 minutes; 1 and 5 mM of Ca2+, Mg2+, Mn2+, Fe3+, Co2+, Cu2+, Zn2+, Ni2+, or Hg2+ and EDTA, Triton X-100 at 5 and 10 mM, and SDS 1%. Neopullulanase activities were measured by soluble starch assay described in EXAMPLE 4. The enzymatic activity without any additive was regarded as control. Relative activity measured as described in example 6.
Additive Relative Activity (%)
ImM 5mM
Figure imgf000016_0001
Industrial Applicability
[0159] The idea can be used in pharmaceutical (e.g. production of cyclodextrin) and food industries,
[0160] In particular, the variants may be used in production of cyclodextrin in which has a wide range of application in complexion materials in foods, pharmaceuticals, plastics, and agricultural products as emulsifiers, antioxidants, and stabilizing agents.
[0161] More particular, the variants may be used in starch processing applications especially, starch saccharification and Liquefaction.
[0162] The present invention also can be used in the production of sweeteners and production ethanol, such as fuel, drinking and industrial ethanol, from starch or whole grains.
Reference Signs List
[0163] The inventors have used methods in the art of genetic engineering in particular site direct mutagenesis to alter the amino acid at position 33 at the sequence ID No. 1 to obtain the sequence ID No. 2, wherein histidin is substitute with glycine, where in the variant has neopullulanase activity; and the variant has at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
[0164] The substrate preference of neopullulanse variant of the present invention was determined as described here in at example 5. Hydrolytic activity was monitored using five different substrates: starch, pullulan, b-cyclodextrin, amylopectin, and glycogen. The optimum enzymatic activity was seen on the pullulan substrate, followed by b-cyclodextrin (68%) and amylopectin (51%). The hydrolytic activity on starch and glycogen was less than 30% of its maximum activity on pullulan.
[0165] The increased productivity of neopullulanase variant was evaluated as described herein at examples of 6-10. The neopullulanase variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 70% relative activity at 60 °C, at least 60% relative activity at 70 °C , at least 50% relative activity at 80 °C when measured at 75° C .
[0166] The neopullulanase variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ~5, at least 100% relative activity at pH ~6, 7 and 8, at least 75% relative activity at pH ~9, at least 70% relative activity at pH ~10. Reference to Deposited Biological Material
[0167] Not applicable.
Sequence Listing Free Text
[0168] Not applicable.
Citation List
[0169] Citation List follows:
[0170] Agaisse, H., & Lereclus, D. (1994). Structural and functional analysis of the promoter region involved in full expression of the crylllA toxin gene of Bacillus thuringiensis. Molecular microbiology, 13(1), 97-107.
[0171] Amann, E., Ochs, B., & Abel, K. J. (1988). Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene, 69(2), 301-315.
Patent Literature
[0172] PTL1 : US patent documents
Figure imgf000018_0001
[0173] Other Patent Documents
Figure imgf000018_0002
Figure imgf000019_0001
Non Patent Literature
[0174] NPL1 : Hue, K. K„ Cohen, S. D„ & Bechhofer, D. H. (1995). A polypurine sequence that acts as a 5'mRNA stabilizer in Bacillus subtilis. Journal of bacteriology, 777(12), 3465-3471.
[0175] NPL2: Simonen, M., & Palva, I. (1993). Protein secretion in Bacillus species. Microbiology and Molecular Biology Reviews, 57(1 ), 109-137.

Claims

Claims
[Claim 1] A neopullulanase variant comprising a substitution at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1 wherein the variant has pullulanase activity; and
(a) Wherein the variant has at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 , and
(b) Wherein the variant has increased thermo-stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 70% relative activity at 60 °C, more particularly, at least 60% relative activity at 70 °C , more particularly at least 50% relative activity at 80 °C when measured at 75° C and,
(c) Wherein the variant has increased pH stability compared to the neopullulanase of SEQ ID NO: 1 , particularly when the variant incubated for 60 minutes has at least 90% relative activity at pH ~5, more particularly, at least 100% relative activity at pH ~6, 7 and 8, more particularly, at least 75% relative activity at pH ~9, more particularly, at least 70% relative activity at pH ~10 when comparing p of SEQ ID NO: 1.
[Claim 2] A method for producing a variant neopullulanase of a parent neopullulanase comprising substitution of the parent neopullulanase at one position corresponding to position 33 of the polypeptide of SEQ ID NO: 1 wherein the variant has pullulanase activity; and wherein the variant has at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
[Claim 3] A use of a variant neopullulanase of claim 1 for production of a syrup and/or a fermentation product, e.g., ethanol, from a starch containing material.
[Claim 4] The use of a neopullulanase variant of claim 1 , wherein the starch material is gelatinized or un-gelatinized starch material.
[Claim 5] A process of producing a fermentation product from starch-containing material by a neopullulanase variant of claim 1 comprising the steps of: (a) Liquefying starch-containing material in the presence of an alpha amylase.
(b) Scarifying the liquefied material in the presence of a glucoamylase; and
(c) Fermenting with a fermenting organism.
[Claim 6] A process of producing a fermentation product from starch-containing material, comprising the steps of:
(a) Saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; by a neopullulanase variant of claim 1 and
(b) Fermenting with a fermenting organism.
[Claim 7] Wherein step (a) is carried out using at least a glucoamylase, and a variant neopullulanase of any of claim 1.
[Claim 8] The process of producing a fermentation product from starch- containing material of claim 6, wherein an alpha amylase is added in step (a).
[Claim 9] The process of producing a fermentation product from starch- containing material of claim 6, wherein saccharification and fermentation is carried out simultaneously.
[Claim 10] The process of producing a fermentation product from starch- containing material of claim 5 wherein the fermentation product is an alcohol, particularly ethanol.
[Claim 11] The process of producing a fermentation product from starch- containing material of claim 6 wherein the fermentation product is an alcohol, particularly ethanol.
[Claim 12] The process of producing a fermentation product from starch- containing material of claim 7 wherein the fermentation product is an alcohol, particularly ethanol.
[Claim 13] The process of producing a fermentation product from starch- containing material of claims 8, wherein the fermentation product is an alcohol, particularly ethanol.
[Claim 14] A polynucleotide encoding the variant neopullulanase of claim 1.
[Claim 15] A nucleic acid constructs comprising the polynucleotide of the variant neopullulanase of claim 1.
[Claim 16] An expression vector comprising the polynucleotide of the variant neopullulanase claiml .
[Claim 17] A host cell comprising the polynucleotide of the variant neopullulanase of claim 1.
[Claim 18] A method of producing a neopullulanase variant of claim 1 , comprising cultivating the host cell of claim 17 under conditions conducive for production of the polypeptide.
[Claim 19] The method of producing a neopullulanase variant of claim 18, further comprising recovering the polypeptide.
[Claim 20] A whole broth formulation or cell culture composition comprising a polypeptide of any of the variant neopullulanase of claim 1.
PCT/IR2021/050010 2021-04-21 2021-04-21 Archaeal neopullulanase variant and increased ph/thermo stability WO2022224279A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011123505A1 (en) * 2010-03-30 2011-10-06 Novozymes North America, Inc. Processes of producing a fermentation product

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011123505A1 (en) * 2010-03-30 2011-10-06 Novozymes North America, Inc. Processes of producing a fermentation product

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 25 November 2020 (2020-11-25), ANONYMOUS : "neupullulanase, partial [synthetic construct] ", XP093000582, retrieved from NCBI Database accession no. QPF70820.1 *

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