A novel cold-active type I pullulanase from a hot-spring metagenome for effective debranching and production of resistant starch

Thakur, Manju Kumari; Sharma, Nitish; Kumar, Amit; Singh, Sudhir P.

doi:10.1016/j.biortech.2020.124288

Cited by 26 publications

(17 citation statements)

References 45 publications

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“…Type 1 pullulanase is a potential biocatalyst that hydrolyzes α-16-linked branches in amylopectin, releasing linear glucan chains. , The linear glucan chains are highly competent for complexation applications and thus can be used for making amylose–fatty acid complexes. In the present study, gelatinized corn starch (GCS) was treated with Pul M type 1 pullulanase, which potentially catalyzed the debranching of starch, leading to a substantial increment (∼60%) in amylose proportion (Figure S1). The debranched corn starch (DCS), containing glucan chain helices, was subjected to fatty acid complexation.…”

Section: Resultsmentioning

confidence: 99%

“…Type 1 pullulanase (Pul M ) was expressed in Escherichia coli BL21 cells, followed by extraction and purification of protein as described in our previous reports. 5,7 2.3. Preparation of Debranched Corn Starch and DCS−Fatty Acid Complex.…”

Section: Expression and Purification Of Type 1 Pullulanase (Pul M )mentioning

confidence: 99%

“…RS-5 refers to the amylose–lipid complexes . RS-3, RS-4, and RS-5 are thermally stable and digestion-resistant to a greater extent, exerting a prebiotic impact on human health . Many studies have demonstrated the physicochemical and functional properties of different resistant starch types …”

Section: Introductionmentioning

confidence: 99%

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Structural Characteristics, Physicochemical Properties, and Digestibility Analysis of Resistant Starch Type-V Prepared from Debranched Corn Starch and Fatty Acid Complexation

Thakur¹,

Kumar

Singh³

2023

ACS Omega

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Corn starch was gelatinized and treated with a metagenomic type 1 pullulanase (Pul M ), increasing the proportion of linear glucan chains. The debranched corn starch (DCS), containing amylose helices, was subjected to complexation with fatty acid molecules at moderate temperatures (50−60 °C). The amylose−lipid complexes prepared using saturated fatty acids, e.g., capric acid (CA) and lauric acid (LA), displayed higher CI values as compared to that of unsaturated fatty acid compounds, e.g., undecylenic acids (UAs) and oleic acid (OA). The DCS−fatty acid complex was estimated to contain about 14% of rapidly digested starch (RDS), 26% of slowly digested starch (SDS), and 60% of resistant starch V (RS-5). RS-5 samples exhibited high resistance toward digestive enzymatic hydrolysis. The surface microdetails of RS-5 were examined by scanning electron microscopy (SEM), depicting small spherulite-like structural aggregates. X-ray diffraction pattern analysis estimated about 46% of the crystallinity of RS-5. Thermal attributes of RS-5 were examined by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis, depicting the increase in melting enthalpies after the complexation of fatty acid molecules with debranched corn starch. Comparative DSC thermograms divulged a relatively higher stability of RS-5 as compared to that of RS-3. The findings advocated the potentiality of RS-5 (nondigestible DCS-LA complex) as a functional, valuable ingredient in the food industry.

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Section: Resultsmentioning

confidence: 99%

Section: Expression and Purification Of Type 1 Pullulanase (Pul M )mentioning

confidence: 99%

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Structural Characteristics, Physicochemical Properties, and Digestibility Analysis of Resistant Starch Type-V Prepared from Debranched Corn Starch and Fatty Acid Complexation

Thakur¹,

Kumar

Singh³

2023

ACS Omega

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“…Combination of enzymes and synergistic interactions between them could develop the bread characteristics and dough rheology. [10] Geobacillus thermocatenulatus DSMZ730 70 °C-pH 6.5 <50% activity after 120 min in 70 °C 7-8 [46] Bacillus methanolicus PB1 50 °C-pH 5.5 <20% activity after 120 min in 60 °C 5.5-6.5 [49] Anoxybacillus SK3-4 60 °C-pH 6 <10% activity after 60 min in 70 °C 5-7 [45] Paenibacillus polymyxa Nws-pp2 35 °C-pH 6 <10% activity after 50 min in 50 °C 5-6.5 [50] Exiguobacterium SH3 45 °C-pH 8.5 <10% activity after 90 min in 70 °C 5-11 [51] Shewanella arctica 35 °C-pH 6-7 <10% activity after 60 min in 40 °C 5-8 [52] Bacillus pseudofirmus 703 45 °C-pH 7-8 <20% activity after 10 min in 50 °C 6.5-8.5 [53] Metagenome-derived 40 °C-pH 6 -6-8 [18] Metagenome-derived (PersiPul1) 60 °C-pH 7 58.70% activity after 120 min in 80 °C 4-9 This study The same findings were reported after the usage of thermostable enzyme cocktail in the wheat bread. [9] 3.3.…”

Section: Synergistic Relationship Of the Persipul1and Persiamy2mentioning

confidence: 99%

“…[13,16,17] Previous studies have shown the usage of metagenomic data from the soil, ruminal fluid, acid mine drainage, fecal microbes, hot springs, and intestines to identify novel 𝛼-amylases and pullulanases. [4,18] There is a growing demand for functional food in recent years associated with a healthy lifestyle. Functional food provides beneficial effects on the human body, improves general health, or/and reduces the risk of diseases.…”

Section: Introductionmentioning

confidence: 99%

Synergistic Effect of Metagenome‐Derived Starch‐Degrading Enzymes on Quality of Functional Bread with Antioxidant Activity

et al. 2021

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Starch-degrading enzymes have gained particular importance in recent decades due to their crucial role in numerous industrial applications especially, in the bakery industry. In this study, a novel thermostable pullulanase (PersiPul1) is screened from the metagenomic data for improving the quality of functional bread. The novel PersiPul1 is active over a wide range of temperature (30-80 °C) and pH (4-9) and exhibits high thermal stability, retaining 58.70% of activity at 80 °C. A cocktail of thermostable pullulanase and 𝜶-amylase (PersiPul1and PersiAmy2) is developed to be used in bread fortified with quinoa protein (Chenopodium quinoa Willd). The bread fortified with 7% quinoa protein isolate possess the highest reducing power and ABTS (73.64%) and DPPH (72.27%) radical scavenging activities. The addition of the enzyme mixture decreases the hardness and chewiness of the bread. The porosity, specific volume, and browning index of bread with pullulanase and 𝜶-amylase are higher than control. Also, sensory analysis of the functional bread reveals higher scores in the presence of enzymes. Based on the results, the novel starch-degrading enzyme cocktail is a promising alternative for improving the physical and sensory characteristics of the antioxidant bread enriched with quinoa protein isolate to be used in the bakery industry as a potential bio-additive.

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Cloning, purification, and functional characterization of recombinant pullulanase from Bacillus cereusATCC 14579 for improved detergent performance

Zafar,

Masood,

Rahman

et al. 2024

J Surfact & Detergents

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The present study outlines the approach that was employed for cloning, expression, and characterization of the recombinant pullulanase enzyme from Bacillus cereus ATCC 14579 into Escherichia coli BL21(DE3) using pET‐25b (+) expression vector. The recombinant pullulanase enzyme was purified using ammonium sulfate precipitation and immobilized metal ion affinity chromatography (IMAC). The molecular mass of the purified pullulanase enzyme was measured using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as 95 kDa. The purified recombinant pullulanase enzyme demonstrated significant thermal stability, maintaining its structural integrity and functionality at temperatures as high as 90°C over a period of 4 h. The inclusion of divalent metal ions, specifically Ca2+ and Mg2+, had a positive effect on the activity of the pullulanase enzyme. Conversely, the presence of Co2+ and EDTA (Ethylene Diamine Tetra Acetic acid) resulted in suppression of the enzyme activity. The purified pullulanase enzyme demonstrated remarkable resistance when exposed to organic solvents. The enzyme activity was notably decreased in the presence of SDS (Sodium Dodecyl Sulfate) while β‐mercaptoethanol and tween‐60 did not substantially affect the enzyme activity and stability which suggest its potential applicability in the detergent sector. This discovery indicates a potential approach to improve the effectiveness of currently available detergents in the marketplace.

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A novel cold-active type I pullulanase from a hot-spring metagenome for effective debranching and production of resistant starch

Cited by 26 publications

References 45 publications

Structural Characteristics, Physicochemical Properties, and Digestibility Analysis of Resistant Starch Type-V Prepared from Debranched Corn Starch and Fatty Acid Complexation

Structural Characteristics, Physicochemical Properties, and Digestibility Analysis of Resistant Starch Type-V Prepared from Debranched Corn Starch and Fatty Acid Complexation

Synergistic Effect of Metagenome‐Derived Starch‐Degrading Enzymes on Quality of Functional Bread with Antioxidant Activity

Cloning, purification, and functional characterization of recombinant pullulanase from Bacillus cereusATCC 14579 for improved detergent performance

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