INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2022
·Vol. 23
·期 13
·6928
·
IF 6.208
Meixing Wang,Huizhen Hu,Buyu Zhang,Yang Zheng,Pan Wu,Zhenghui Lu,Guimin Zhang
Panose is a type of functional sugar with diverse bioactivities. The enzymatic conversion bioprocess to produce high purity panose with high efficiency has become increasingly important. Here, a new neopullulanase (NPase), Amy117 fromB. pseudofirmus703, was identified and characterized. Amy117 presented the optimal activity at pH 7.0 and 30 °C, its activity is over 40% at 10 °C and over 80% at 20 °C, which is cold-active. The enzyme cleaved α-1, 4-glycosidic linkages of pullulan to generate panose as the only hydrolysis product, and degraded cyclodextrins (CDs) and starch to glucose and maltose, with an apparent preference for CDs. Furthermore, Amy117 can produce 72.7 mg/mL panose with a conversion yield of 91% (w/w) based on 80 mg/mL pullulan. The sequence and structure analysis showed that the low proportion of Arg, high proportion of Asn and Gln, and high α-helix levels in Amy117 may contribute to its cold-active properties. Root mean square deviation (RMSD) analysis also showed that Amy117 is more flexible than two mesophilic homologues. Hence, we discovered a new high-efficiency panose-producing NPase, which so far achieves the highest panose production and would be an ideal candidate in the food industry.Keywords:neopullulanase (NPase);pullulan;panose production;high-efficiency
研究领域:
食品科学
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酶学
·
生物技术
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产品关联
DOI
PubMed
高影响因子
Food Bioscience
·
2024
·
IF 4.8
Yue Dai,Xiaoxiao Li,Zhengyu Jin,Yuxiang Bai
Improving substrate utilization is crucial for industrial conversion of starch. The introduction of pullulanase in cyclodextrin (CD) production has improved substrate utilization. However, complete utilization of debranched starches has not been achieved, mainly due to the retrogradation of debranched starches and the inhibitory effect of CDs on pullulanase during the catalytic reaction. This study aimed to establish an efficient CD preparation system by regulating the substrate concentration and reaction temperature. A Bacillus thermoleovorans pullulanase (BtPul) was selected for investigation of its interaction with CDs through inhibition pattern assays, fluorescence spectroscopy, and molecular docking. Results clarified that CDs inhibited BtPul, with α-CD showing the highest inhibition, followed by β-CD and γ-CD. γ-CD inhibited BtPul through a mixed mode of competitive and noncompetitive inhibition, while the other CDs inhibited BtPul competitively. When pullulan was used as the substrate, increasing the substrate concentration weakened the inhibitory effect of CDs to some degree, while changes in reaction temperature only had a minor contribution. When BtPul and β-CGTase collaboratively converted 30% (w/v) starch substrate, an increase in reaction temperature improved the conversion rate of β-CD from 67.6% to 86.8%. Optimization of substrate concentration showed that high concentration starch substrate had an adverse effect on CDs yield due to retrogradation. The optimal yields of α-CD and β-CD produced by BtPul and α-/β-CGTases were 55.7% and 90.3%, respectively. This study provides a reliable method and reference for the efficient conversion of starch into CDs.
研究领域:
生物化学
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食品科学
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工业微生物学
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产品关联
DOI
高影响因子
FOOD HYDROCOLLOIDS
·
2021
·
IF 9.147
Junyu Tang,Jianwei Zhou,Xinyue Zhou,Dandan Li,Zhengzong Wu,Jinhu Tian,Enbo Xu,Donghong Liu
Resistant starch (RS) is widely used as a new type of dietary fiber, but it is still a limitation of controllable formation of microstructure and polymeric architecture for preparing single-component RS (pure-phase). In this study, RS was prepared using maize starch with two ratios of amylose to amylopectin (RAA 1:1 and RAA 1:2 ) and four pullulanase concentrations (5, 15, 25, 35 U/g) in a high-solid dynamic reaction environment of bio-extrusion. Based on the long- and short-range orders reflected by the glycosidic bonds ( 13 C NMR), chain-length distributions (SEC/FACE), single-/double-helices (FTIR), lamellas (SAXS) and crystalline pattern (XRD), the rearranged supramolecular structure of starch and their relationships with physicochemical properties (DSC and RVA) was illuminated. There were two morphologies of RS were created: 1) disordered fusion-like structure displayed by RAA 1:1 -P15 and RAA 1:2 -P35, which had the high contents of RS (54.95 and 54.19%) and short amylopectin (DP ≤ 24) (70.52% and 60.20%). The short (A chains) and extremely short (DP < 6) amylopectin could contribute to form submicro-crystallite with B1 chains in the extruded matrix at a low moisture level; 2) thinner, scalier and layered-like structure displayed by RAA 1:2 -P15 that was the transition state from thick layered strips of large granules. Its SDS content was high (33.61%) and owned the larger rearranged d Lorentz (16.97 nm). Overall, the entangled regime of amylose and amylopectin in the modifications of starch with different structural units or micro-domains in this study will provide a new idea and reference to explore the preparation of novel RS.
研究领域:
食品科学
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生物化学
✓ 已验证
DOI
高影响因子
PROCESS BIOCHEMISTRY
·
2020
·
IF 2.952
Han Huang,Yun Lin,Guozeng Wang,Juan Lin
A new pullulanase-encoding gene, pulSL3 , was cloned from Alkalibacterium sp. SL3 and expressed in Escherichia coli . Deduced PulSL3 contains 2026 amino acid residues and consists of a putative signal peptide, four carbohydrate-binding modules, an E_set pullulanase domain, an α-amylase domain, and a pullulanase domain. The purified rPulSL3 exhibited pullulanase activity only. To identify the functions of the α-amylase and pullulanase domains, the N- and C-terminus truncated mutants ( pulSL3△N and pulSL3△C ) were also expressed in E. coli. The purified rPulSL3△N had pullulanase activity, while rPulSL3△C had no either α-amylase or pullulanase activity. Both rPulSL3 and rPulSL3△N exhibited maximum activities at pH 9.0 and 50 °C, and were highly active and stable at the concentration of NaCl up to 4 M and resistant to a few surfactants and detergents. In comparison to the wild type, rPulSL3△N had better thermostability (retaining 62.9 vs. 33.6% activity at 52 °C for 30 min), higher substrate affinity (with K m of 0.10 vs. 0.15 mg mL −1 ) and higher catalytic efficiency (836.6 vs. 520.3 mL mg −1 s −1 ). These excellent properties make rPulSL3△N potential for the application in the industries of detergent, food, and environmental remediation.
研究领域:
微生物学
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酶学
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生物技术
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产品关联
DOI
高影响因子
Foods
·
2022
·Vol. 11
·期 23
·3779
·
IF 5.561
David Mahoudjro Bodjrenou,Xin Li,Wei Chen,Yi Zhang,Baodong Zheng,Hongliang Zeng
The effects of pullulanase debranching combined with autoclaving (PDA) at various debranching times (0 h, 5 h, 10 h, 15 h, 20 h, and 25 h) and 121 °C/20 min of autoclave treatment on the structural and physicochemical characteristics of purple sweet potato (Jinshu No.17) starch were investigated. The results indicated that the native starch (NS) was polygonal, round, and bell-shaped with smooth surfaces. After debranching treatment, the surface of the starch samples became rough and irregular. The molecular weight became smaller after treatments. X-ray diffraction C-type pattern was transformed into a B-type structure in treated samples with increased relative crystallinity.13C NMR indicated an increased propensity for double helix formation and new shift at C1, 3, 5 region compared to NS. The apparent amylose content was 21.53% in the NS. As the swelling power decreased, the percentage of soluble solids increased and different thermal properties were observed. A higher yield of the resistant starch (RS) was observed in all treated starch except PDA 25 h. The findings of our study reveal that a combination of pullulanase debranching time (15 h) and autoclaving (121 °C for 20 min) is a great technique that can be used to produce a higher amount of resistant starch in the Jinshu No.17 starch.Keywords:purple sweet potato resistant starch;pullulanase debranching;autoclave treatment;structural characteristics;physicochemical properties
研究领域:
食品科学
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淀粉化学
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生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2017
·
IF 4.529
Jie Long,Bao Zhang,Xingfei Li,Xiaobei Zhan,Xueming Xu,Zhengjun Xie,Zhengyu Jin
In this study, pullulanase was firstly immobilized by covalent bonding onto chitosan/Fe 3 O 4 nanoparticles or encapsulation in sol-gel after bonding onto chitosan/Fe 3 O 4 nanoparticles, and then the immobilized pullulanase was used for the effective production of resistant starch (RS). The highest RS content (35.1%) was obtained under the optimized condition of pH 4.4, enzyme concentration of 10 ASPU/g and hydrolysis time of 12 h when debranched by free pullulsanase, indicating that RS content was significantly (p < 0.05) increased when compared to native starch (4.3%) and autoclaved starch (12.5%). Under these conditions, the immobilized pullulanase (10 ASPU/g dry starch) yielded higher RS content compared to free enzyme (10 ASPU/g dry starch), especially, the pullulanse immobilized by sol-gel encapsulation yielded the highest RS content (43.4%). Moreover, compared to starches hydrolyzed by free pullulanase, starches hydrolyzed by immobilized pullulanase showed a different saccharide profile of starch hydrolysate, including a stronger peak C (MW = 5.0 × 10 3 ), as well as exhibited an additional absorption peak around 140 °C. Reusability results demonstrated that pullulanase immobilized by sol-gel encapsulation had the advantages of producing higher RS content as well as better operational stability compared to pullulanase immobilized by cross-linking. The resulting enhanced RS content generated by the process described in this work could be used as an adjunct in food processing industries.
研究领域:
食品科学
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生物化学
·
酶工程
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产品关联
DOI
PubMed
高影响因子
Foods
·
2022
·Vol. 11
·期 10
·1442
·
IF 5.561
Yamei Wang,Yixiong Tian,Xiaofeng Ban,Caiming Li,Yan Hong,Li Cheng,Zhengbiao Gu,Zhaofeng Li
Amylo-α-1,6-glucosidase (EC 3.2.1.33, AMY) exhibits hydrolytic activity towards α-1,6-glycosidic bonds of branched substrates. The debranching products of maltodextrin, waxy corn starch and cassava starch treated with AMY, pullulanase (EC 3.2.1.41, PUL) and isoamylase (EC 3.2.1.68, ISO), were investigated and their differences in substrate selectivity and debranching efficiency were compared. AMY had a preference for the branched structure with medium-length chains, and the optimal debranching length was DP 13–24. Its optimum debranching length was shorter than ISO (DP 25–36). In addition, the debranching rate of maltodextrin treated by AMY for 6 h was 80%, which was 20% higher than that of ISO. AMY could decompose most of the polymerized amylopectin in maltodextrin into short amylose and oligosaccharides, while it could only decompose the polymerized amylopectin in starch into branched glucan chains and long amylose. Furthermore, the successive use of AMY and β-amylase increased the hydrolysis rate of maltodextrin from 68% to 86%. Therefore, AMY with high substrate selectivity and a high catalytic capacity could be used synergistically with other enzyme preparations to improve substrate utilization and reduce reaction time. Importantly, the development of a novel AMY provides an effective choice to meet different production requirements.Keywords:amylo-α-1,6-glucosidase;substrate selectivity;pullulanase;isoamylase;debranching efficiency
研究领域:
生物化学
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酶学
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食品科学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2020
·
IF 5.162
Xiong Lin,Siwei Sun,Bailong Wang,Baodong Zheng,Zebin Guo
Lotus seed starch (LS) was enzymatically hydrolyzed by pullulanase, β-amylase and α-amylase to obtain three types of starch nanoparticles (SNPs): P-SNPs, β-SNPs and α-SNPs, respectively. The structure and physicochemical properties of lotus seed starch nanoparticles (LS-SNPs) were systematically studied by Laser particle size analysis, Scanning electron microscope (SEM), X-ray diffraction (XRD), Raman spectrometry, Nuclear magnetic resonance (NMR) and Gel permeation chromatography (GPC). The results showed that compared to LS (10,466.7 ± 230.9 nm), the D [3,2] of P-SNPs were (334.7 ± 16.2), which indicated that LS was decomposed into LS-SNPs with low degree of polymerization. P-SNPs showed the highest efficiency, with 81.74% of samples reaching the nanoscale. The particle size order was LS > β-SNPs (α-SNPs) > P-SNPs. XRD results showed P-SNPs had the highest crystallinity 65.07%. During preparation of LS-SNPs, crystal region was destroyed, and double helix structure became stronger, but no new functional groups were introduced. P-SNPs had the smallest amorphous area and also showed a minimal molecular weight and the broadest molecular weight distribution. P-SNPs had the smallest particle size and the highest crystallinity, so P-SNPs were the best of all LS-SNPs.
研究领域:
食品科学
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材料科学
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化学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2019
·
IF 4.784
Xiaoxiao Li,Yuxiang Bai,Hangyan Ji,Jinpeng Wang,Yinglu Cui,Zhengyu Jin
Pullulanase, a typical debranching enzyme, specifically hydrolyzes α-1,6 glycosidic linkages in pullulan, starch and so on. There is accumulated knowledge about the catalysis of pullulanase, but functional roles of acceptor subsites get less attention. According to the crystal structure of pullulanase from Bacillus subtilis str. 168, stacking interaction between tryptophan437 and glycosyl unit of α-CD was found. Trp437 located in conserved region III was highly conserved in pullulanases. But functional role of this residue remains unclear. Site-directed mutagenesis was used to determine the function of Trp437. Replacement of Trp437 with glycine, phenylalanine, proline, arginine resulted in mutants with undetectable hydrolysis activity and reverse hydrolysis activity. The secondary structure of mutated proteins showed no difference from the wild-type pullulanase, but they lost the capability to bind β-CD based on the ITC measurement. Molecular docking was performed to investigate the affinity of proteins to ligands including maltotriose, 6 2 -α-D-maltotriosyl-maltotriose and α-CD, showing the affinity of mutants to ligands became weaker compared to that of PulA. And the hydrogen bond between Trp437 and Glu525 in PulA only was found in mutant W437R. These results show that Trp437 at subsite +2 plays crucial role in catalysis of PulA, and it has little tolerance to change.
研究领域:
酶学
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微生物学
·
结构生物学
✓ 已验证
DOI
PubMed
高影响因子
JOURNAL OF BIOTECHNOLOGY
·
2020
·
IF 3.503
Shi-Yu Zhang,Ze-Wang Guo,Xiao-Ling Wu,Xiao-Yang Ou,Min-Hua Zong,Wen-Yong Lou
Cold-adapted pullulanase with high catalytic activity and stability is of special interest for its wide application in cold starch hydrolysis, but few pullulanases displaying excellent characteristics at ambient temperature and acidic pH have hitherto been reported. Here, a novel pullulanase from Bacillus methanolicus PB1 was successfully expressed in Escherichia coli BL21 (DE3) and determined to be a cold-adapted type I pullulanase (PulPB1) with maximum activity at 50 °C and pH 5.5. The recombinant PulPB1 showed great stability, its half-life at 50 °C was 137 h. PulPB1 can efficiently hydrolyze pullulan and amylopectin, with activities of 292 and 184 U/mg at 50 °C and pH 5.5, respectively. Moreover, the N-terminal domain of PulPB1 was found to significantly affect the enzymatic performance. Following truncation of the N-terminal domain, the activity towards pullulan decreased markedly from 292 to 141 U/mg and the half-life at 50 °C decreased from 137 to 10 h. Compared to the hydrolysis system with amyloglucosidase alone, the catalytic efficiency showed a 2.4-fold increase on combining PulPB1 with amyloglucosidase for amylopectin hydrolysis at 40 °C. This demonstrates that PulPB1 is promising for development as a superior candidate for cold amylopectin hydrolysis.
研究领域:
生物化学
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微生物学
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酶学
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生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2024
·
IF 8.5
Ting Xie,Li Zhou.,Laichuang Han,Cuiping You,Zhongmei Liu,Wenjing Cui,Zhongyi Cheng,Junling Guo,Zhemin Zhou
Pullulanase is a starch-debranching enzyme that hydrolyzes side chain of starch, oligosaccharides and pullulan. Nevertheless, the limited activities of pullulanases constrain their practical application. Herein, the hyperthermophilic type II pullulanase from Pyrococcus yayanosii CH1 (Pul PY2 ) was evolved by synergistically engineering the substrate-binding pocket and active-site lids. The resulting mutant Pul PY2 -M2 exhibited 5-fold improvement in catalytic efficiency ( k cat / K m ) compared to that of Pul PY2 . Pul PY2 -M2 was utilized to develop a one-pot reaction system for efficient production of maltooligosaccharides. The maltooligosaccharides conversion rate of Pul PY2 -M2 reached 96.1%, which was increased by 5.4% compared to that of Pul PY2 . Furthermore, when employed for glucose production, the glucose productivity of Pul PY2 -M2 was 25.4% and 43.5% higher than that of Pul PY2 and the traditional method, respectively. These significant improvements in maltooligosaccharides and glucose production and the efficient utilization of corn starch demonstrated the potential of the engineered Pul PY2 -M2 in starch sugar industry.
研究领域:
生物化学
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生物技术
·
酶学
·
食品科学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF FOOD BIOCHEMISTRY
·
2019
·Vol. 43
·期 8
·e12890
·
IF 1.358
Yuxiang Bai,Yun Wu,Hangyan Ji,Zhengyu Jin
Glucosyl-β-cyclodextrin (G 1 -β-CD) has wide application prospects in the food, pharmaceutical, agriculture, and chemical industry as its better solubility and biocompatibility than parent β-cyclodextrin (β-CD). However, researches on G 1 -β-CD were few due to difficulties in its preparation and separation. In the present study, a one-pot method was as follows: first, maltosyl-β-cyclodextrin (G 2 -β-CD) was prepared through the reverse synthesis capability of pullulanase. Then, G 1 -β-CD was obtained through hydrolysis by glucoamylase in the same reaction system and further purified by gel filtration chromatography. The results revealed that the optimal conditions for the reverse synthesis were as follows: n G2 :n β-CD , 8:1; enzyme concentration, 60 U/ml; reaction temperature, 60°C; pH, 4.5; reaction time, 60 hr. The optimal conditions for G 2 -β-CD hydrolysis were as follows: reaction time, 8 hr; enzyme concentration, 40 U/ml. Finally, the pure G 1 -β-CD samples were obtained by Sephadex G-25 gel column separation and identified by HPLC, LC-MS/MS, and NMR chromatography. Practical applications Commercially available modified CDs are almost synthesized using the chemical method, such as methyl-, sulfonylbutyl-, and hydroxypropyl-CD. However, the chemical synthesis of modified CDs has some security issue, such as organic solvent residue, which limits its application in foods. Based on the relevance references, G1-β-CD not only has the inclusion properties of the parent β-CD but also improves solubility and reduces the hemolysis effect and nephrotoxicity. However, the synthesis and separation of G1-β-CD is an obstacle to its expansion in production and application. In addition, there are few studies on G1-β-CD and few companies selling the product on the market. In this manuscript, the G1-β-CD was successfully synthesized by one-pot method and the optimal preparation and separation conditions of G1-β-CD were investigated.
研究领域:
食品科学
·
药学
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化学工程
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农业科学
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）