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12 pages, 1777 KiB

Open AccessArticle

Efficient Utilization of Fruit Peels for the Bioproduction of D-Allulose and D-Mannitol

by Jin Li, Jiajun Chen, Wei Xu, Wenli Zhang, Yeming Chen and Wanmeng Mu

Foods 2022, 11(22), 3613; https://doi.org/10.3390/foods11223613 - 12 Nov 2022

Cited by 4 | Viewed by 2863

Currently, the demand for low-calorie sweeteners has grown dramatically because consumers are more mindful of their health than they used to be. Therefore, bioproduction of low-calorie sweeteners from low-cost raw materials becomes a hot spot. In this study, a two-stage strategy was established [...] Read more.

Currently, the demand for low-calorie sweeteners has grown dramatically because consumers are more mindful of their health than they used to be. Therefore, bioproduction of low-calorie sweeteners from low-cost raw materials becomes a hot spot. In this study, a two-stage strategy was established to efficiently utilize D-fructose from fruit and vegetable wastes. Firstly, ketose 3-epimerase was used to produce D-allulose from D-fructose of pear peels. Secondly, the residual D-fructose was converted to D-mannitol by the engineered strain co-expression of D-mannitol 2-dehydrogenase and formate dehydrogenase. Approximately 29.4% D-fructose of pear peels was converted to D-allulose. Subsequently, under optimal conditions (35 °C, pH 6.5, 1 mM Mn²⁺, 2 g/L dry cells), almost all the residual D-fructose was transformed into D-mannitol with a 93.5% conversion rate. Eventually, from 1 kg fresh pear peel, it could produce 10.8 g of D-allulose and 24.6 g of D-mannitol. This bioprocess strategy provides a vital method to biosynthesize high-value functional sugars from low-cost biomass. Full article

(This article belongs to the Special Issue Application of Enzyme Engineering in Food)

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12 pages, 1189 KiB

Open AccessArticle

Preparation of 2-Arachidonoylglycerol by Enzymatic Alcoholysis: Effects of Solvent and Water Activity on Acyl Migration

by Xiaohan Wang, Keying Liu, Yifan Wang, Zhuoneng Huang and Xiaosan Wang

Foods 2022, 11(20), 3213; https://doi.org/10.3390/foods11203213 - 14 Oct 2022

Cited by 6 | Viewed by 1707

Abstract

Enzymatic alcoholysis was performed in an organic medium to synthesize 2-monoacylglycerol (2-MAG) rich in arachidonic acid. The results showed that solvent type and water activity (a_w) significantly affected the 2-MAG yield. Under the optimum conditions, 33.58% 2-MAG was produced in [...] Read more.

Enzymatic alcoholysis was performed in an organic medium to synthesize 2-monoacylglycerol (2-MAG) rich in arachidonic acid. The results showed that solvent type and water activity (a_w) significantly affected the 2-MAG yield. Under the optimum conditions, 33.58% 2-MAG was produced in the crude product in t-butanol system. Highly pure 2-MAG was obtained after two-stage extraction using 85% ethanol aqueous solution and hexane at first stage and dichloromethane and water at second stage. Isolated 2-MAG was used as substrate to investigate the effect of solvent type and a_w on 2-MAG acyl migration in a lipase-inactivated system. The results indicated that non-polar solvents accelerated the acyl migration of 2-MAG, whereas isomerization was inhibited in polar solvent systems. The a_w exhibited the strongest inhibition effect on 2-MAG isomerization at 0.97, but also affected the hydrolysis of glycerides and lipase selectivity. Full article

(This article belongs to the Special Issue Application of Enzyme Engineering in Food)

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13 pages, 3820 KiB

Open AccessArticle

Isolation and Mechanistic Characterization of a Novel Zearalenone-Degrading Enzyme

by Jian Ji, Jian Yu, Wei Xu, Yi Zheng, Yinzhi Zhang and Xiulan Sun

Foods 2022, 11(18), 2908; https://doi.org/10.3390/foods11182908 - 19 Sep 2022

Cited by 10 | Viewed by 2078

Abstract

Zearalenone (ZEN) and its derivatives pose a serious threat to global food quality and animal health. The use of enzymes to degrade mycotoxins has become a popular method to counter this threat. In this study, Aspergillus niger ZEN-S-FS10 extracellular enzyme solution with ZEN-degrading [...] Read more.

Zearalenone (ZEN) and its derivatives pose a serious threat to global food quality and animal health. The use of enzymes to degrade mycotoxins has become a popular method to counter this threat. In this study, Aspergillus niger ZEN-S-FS10 extracellular enzyme solution with ZEN-degrading effect was separated and purified to prepare the biological enzyme, FSZ, that can degrade ZEN. The degradation rate of FSZ to ZEN was 75–80% (pH = 7.0, 28 °C). FSZ can function in a temperature range of 28–38 °C and pH range of 2.0–7.0 and can also degrade ZEN derivatives (α-ZAL, β-ZOL, and ZAN). According to the enzyme kinetics fitting, ZEN has a high degradation rate. FSZ can degrade ZEN in real samples of corn flour. FSZ can be obtained stably and repeatedly from the original strain. One ZEN degradation product was isolated: FSZ−P(C₁₈H26O₄), with a relative molecular weight of 306.18 g/mol. Amino-acid-sequencing analysis revealed that FSZ is a novel enzyme (homology < 10%). According to the results of molecular docking, ZEN and ZAN can utilize their end-terminal carbonyl groups to bind FSZ residues PHE307, THR55, and GLU129 for a high-degradation rate. However, α-ZAL and β-ZOL instead contain hydroxyl groups that would prevent binding to GLU129; thus, the degradation rate is low for these derivatives. Full article

(This article belongs to the Special Issue Application of Enzyme Engineering in Food)

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15 pages, 3628 KiB

Open AccessArticle

Improving the Specific Activity and Thermostability of Psychrophilic Xylosidase AX543 by Comparative Mutagenesis

by Kungang Pan, Zhongqi Liu, Zhengjie Zhang, Shanzheng Jin, Zhao Yu, Tianhui Liu, Tongcun Zhang, Junqi Zhao and Zhongyuan Li

Foods 2022, 11(16), 2463; https://doi.org/10.3390/foods11162463 - 16 Aug 2022

Cited by 3 | Viewed by 1718

Abstract

Improving the specific activity and thermostability of psychrophilic xylosidase is important for improving its enzymatic performance and promoting its industrial application. Herein, a psychrophilic xylosidase AX543 exhibited activity in the temperature range between 0 and 35 °C, with optimum activity at 20 °C, [...] Read more.

Improving the specific activity and thermostability of psychrophilic xylosidase is important for improving its enzymatic performance and promoting its industrial application. Herein, a psychrophilic xylosidase AX543 exhibited activity in the temperature range between 0 and 35 °C, with optimum activity at 20 °C, which is lower than that of other reported psychrophilic xylosidases. The thermostability, specific activity, and catalytic efficiency of the site-directed variants G110S, Q201R, and L2 were significantly enhanced, without affecting the optimal reaction temperature. Comparative protein structural analysis and molecular dynamics simulation indicated that these improvements might be the result of the increased hydrogen bonds interaction and improved structural rigidity. Furthermore, homologous module substitution with four segments demonstrated that the psychrophilic characteristics of AX543 are the results of the whole protein structure, and the C-terminal segment A4 appears to be more essential in determining psychrophilic characteristics, exhibiting potentiality to produce more psychrophilic xylosidases. This study provides valuable structural information on psychrophilic xylosidases and also offers attractive modification strategies to modify catalytic activity, thermostability, and optimal reaction temperature. Full article

(This article belongs to the Special Issue Application of Enzyme Engineering in Food)

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12 pages, 1434 KiB

Open AccessArticle

Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu Apples

by Bin Liu, Xianfang Zhou, Haiyan Guan, Xuequn Pang and Zhaoqi Zhang

Foods 2022, 11(12), 1790; https://doi.org/10.3390/foods11121790 - 17 Jun 2022

Cited by 6 | Viewed by 2195

Abstract

The distinct dark-red skin of Huaniu apples renders them attractive to customers. However, the mechanism that leads to the development of the color of the fruit is unclear. In this study, we found that compared with red Fuji (a bright-red apple cultivar), Huaniu [...] Read more.

The distinct dark-red skin of Huaniu apples renders them attractive to customers. However, the mechanism that leads to the development of the color of the fruit is unclear. In this study, we found that compared with red Fuji (a bright-red apple cultivar), Huaniu apples had higher contents of (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-gallocatechin gallate (GCG), and procyanidins (PCs) B2 and C1 in the peel, which implies that the polymerization of the flavanols and PCs may be correlated with the dark-red skin of the fruit. Using EC as a substrate, we purified an enzyme from Huaniu peel. We performed protein sequencing and discovered that the enzyme was a polyphenol oxidase (PPO). The molecular weight of the enzyme was approximately 140 kDa, which we estimated by native-PAGE and SDS-PAGE, while it was 61 kDa by urea-SDS-PAGE, from which we discovered that the PPO was a dimer. We observed the lowest K_m value for catechol (0.60 mM), and the best substrate was 4-methylcatechol, with a V_max of 526.32 U mg⁻¹ protein. EC is a suitable natural substrate, with a K_m value of 1.17 mM, and 55.27% of the V_max/K_m of 4-methylcatechol. When we used EC as a substrate, the optimum temperature and pH of the PPO were 25 °C and 5.0, respectively. In summary, we purified a dimeric PPO from Huaniu apples that showed high activity to EC, which might catalyze the polymerization of flavanols and PCs and lead to the dark-red color development of the fruit. Full article

(This article belongs to the Special Issue Application of Enzyme Engineering in Food)

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11 pages, 2068 KiB

Open AccessArticle

Enhanced Soluble Expression of Linoleic Acid Isomerase by Coordinated Regulation of Promoter and Fusion Tag in Escherichia coli

by Baixi Zhang, Tong Zhu and Xintian Huang

Foods 2022, 11(10), 1515; https://doi.org/10.3390/foods11101515 - 23 May 2022

Cited by 2 | Viewed by 2410

Abstract

PAI is a linoleic acid isomerase from Propionibacterium acnes and is the key enzyme in the synthesis of trans10, cis12-conjugated linoleic acid. However, the majority of the expressed PAI in Escherichia coli occurs in its nonfunctional form in inclusion bodies, limiting [...] Read more.

PAI is a linoleic acid isomerase from Propionibacterium acnes and is the key enzyme in the synthesis of trans10, cis12-conjugated linoleic acid. However, the majority of the expressed PAI in Escherichia coli occurs in its nonfunctional form in inclusion bodies, limiting the biosynthesis of conjugated linoleic acid. In an attempt to improve the solubility of recombinant PAI in Escherichia coli, three promoters representing different transcriptional strengths (T7, CspA, and Trc), paired with three fusion tags, (His6, MBP, and Fh8), respectively, were investigated in this study. Among the nine recombinant strains, Escherichia coli BL21 (DE3) (pET24a-Mpai), containing the T7 promoter and MBP fusion tag, led to a considerable increase in PAI solubility to 86.2%. MBP-PAI was purified 41-fold using affinity column chromatography. The optimum catalytical conditions of MBP-PAI were 37 °C and pH 7.5 with the addition of 1 mmol/L Tween-20. Most of the tested metal ions inhibited MBP-PAI activity. The apparent kinetic parameters (Km and Vmax) were measured with linoleic acid concentrations ranging from 71 μM to 1428 μM. The substrate linoleic acid did not exert any inhibitory effect on MBP-PAI. The Km of MBP-PAI was 253.9 μmol/L, and the Vmax was 2253 nmol/min/mg. This study provided a new method for improving the solubility of the recombinant linoleic acid isomerase in Escherichia coli. Full article

(This article belongs to the Special Issue Application of Enzyme Engineering in Food)

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13 pages, 1990 KiB

Open AccessArticle

Substrate Selectivity of a Novel Amylo-α-1,6-glucosidase from Thermococcus gammatolerans STB12

by Yamei Wang, Yixiong Tian, Xiaofeng Ban, Caiming Li, Yan Hong, Li Cheng, Zhengbiao Gu and Zhaofeng Li

Foods 2022, 11(10), 1442; https://doi.org/10.3390/foods11101442 - 16 May 2022

Cited by 4 | Viewed by 2572

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Application of Enzyme Engineering in Food)

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14 pages, 3782 KiB

Open AccessArticle

Disulfide Bond Engineering for Enhancing the Thermostability of the Maltotetraose-Forming Amylase from Pseudomonas saccharophila STB07

by Yinglan Wang, Caiming Li, Xiaofeng Ban, Zhengbiao Gu, Yan Hong, Li Cheng and Zhaofeng Li

Foods 2022, 11(9), 1207; https://doi.org/10.3390/foods11091207 - 21 Apr 2022

Cited by 15 | Viewed by 2666

Abstract

Maltooligosaccharides are a novel type of functional oligosaccharides with potential applications in food processing and can be produced by glycosyl hydrolases hydrolyzing starch. However, the main obstacle in industrial applications is the balance between the high temperature of the process and the stability [...] Read more.

Maltooligosaccharides are a novel type of functional oligosaccharides with potential applications in food processing and can be produced by glycosyl hydrolases hydrolyzing starch. However, the main obstacle in industrial applications is the balance between the high temperature of the process and the stability of enzymes. In this study, based on the structural information and in silico tools (DSDBASE-MODIP, Disulfide by Design2 and FoldX), two disulfide bond mutants (A211C-S214C and S409C-Q412C) of maltotetraose-forming amylase from Pseudomonas saccharophila STB07 (MFA_ps) were generated to improve its thermostability. The mutation A211C-S214C was closer to the catalytic center and showed significantly improved thermostability with a 2.6-fold improved half-life at 60 °C and the thermal transition mid-point increased by 1.6 °C, compared to the wild-type. However, the thermostability of mutant S409C-Q412C, whose mutation sites are closely to CBM20, did not change observably. Molecular dynamics simulations revealed that both disulfide bonds A211C-S214C and S409C-Q412C rigidified the overall structure of MFA_ps, however, the impact on thermostability depends on the position and distance from the catalytic center. Full article

(This article belongs to the Special Issue Application of Enzyme Engineering in Food)

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16 pages, 3394 KiB

Open AccessArticle

A Novel Thermal-Activated β-Galactosidase from Bacillus aryabhattai GEL-09 for Lactose Hydrolysis in Milk

by Shuyue Luan and Xuguo Duan

Foods 2022, 11(3), 372; https://doi.org/10.3390/foods11030372 - 27 Jan 2022

Cited by 17 | Viewed by 4305

Abstract

β-Galactosidase has been greatly used in the dairy industry. This study investigated a novel thermostable β-galactosidase (lacZBa) from Bacillus aryabhattai GEL-09 and evaluated the hydrolytic performance of this enzyme. Firstly, the lacZBa-encoding gene was cloned and overexpressed in Escherichia coli BL21(DE3). Phylogenetic analyses [...] Read more.

β-Galactosidase has been greatly used in the dairy industry. This study investigated a novel thermostable β-galactosidase (lacZBa) from Bacillus aryabhattai GEL-09 and evaluated the hydrolytic performance of this enzyme. Firstly, the lacZBa-encoding gene was cloned and overexpressed in Escherichia coli BL21(DE3). Phylogenetic analyses revealed that lacZBa belonged to the glycoside hydrolase family 42. Using SDS-PAGE, we determined that the molecular weight of lacZBa was ~75 kDa. Purified lacZBa exhibited a maximum activity at 45 °C, pH 6.0, and could be activated following incubation at 45 °C for several minutes. The half-life of lacZBa at 45 °C and 50 °C was 264 h and 36 h, respectively. While Co²⁺, Mn²⁺, Zn²⁺, Fe²⁺, Mg²⁺, and Ca²⁺ enhanced enzymatic activity, Cu²⁺ and ethylenediaminetetraacetic acid inhibited enzymatic activity. Moreover, lacZBa could hydrolyze lactose and oNPG with K_m values of 85.09 and 14.38 mM. Molecular docking results revealed that lacZBa efficiently recognized and catalyzed lactose. Additionally, the hydrolysis of lactose by lacZBa was studied in lactose solution and commercial milk. Lactose was completely hydrolyzed within 4 h with 8 U/mL of lacZBa at 45 °C. These results suggested that lacZBa identified in this study has potential applications in the dairy industry. Full article

(This article belongs to the Special Issue Application of Enzyme Engineering in Food)

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