Effect of an endo-1,4-ß-xylanase from wheat malt on water-unextractable arabinoxylan derived from wheat.

BACKGROUND: Non-starch polysaccharides in wheat are dominated by arabinoxylan (AX). Endo-1,4-ß-xylanase (EC 3.2.1.8) is the most important enzyme for degrading AX. This paper investigated the ability of endo-1,4-ß-xylanase extracted from wheat malt to degrade non-water-extractable wheat-derived arabinoxylan (WUAX). RESULTS: The enzyme was observed to break down wheat-derived WUAX effectively, substantially increasing the concentration of water-extractable arabinoxylan (WEAX) in the system for up to 6 h. A considerable quantity of arabinose xylooligosaccharide (AXOS) was also produced, suggesting that the enzyme could produce oligosaccharides too. The molecular weight of the product WEAX was between 23 and 27 kDa and the content of oligosaccharides changed with degradation time. This suggests that endo-1,4-ß-xylanase can not only degrade WUAX into WEAX and xylooligosaccharides but can also degrade the xylooligosaccharides with larger molecular weights into xylobiose and xylotriose. The viscosity of the degradation product increased significantly in the first 2 h, then decreased with longer degradation times. The concentration of WEAX in the reaction system increased throughout the reaction but at gradually lower rates, indicating that the endo-1,4-ß-xylanase degraded WEAX better than it degraded WUAX. Rheological tests showed that solutions prepared from the WEAX that was produced had properties of a pseudoplastic fluid. CONCLUSION: The results showed that the wheat malt endo-1,4-ß-xylanase, which we had previously tested on WEAX, was also effective in degrading wheat-derived WUAX. This study can therefore provide a theoretical basis for the subsequent role of the enzyme in other sources of xylan, and provide guidance for the quality control of beer in the brewing process. © 2021 Society of Chemical Industry.

Purification, Identification, and Characterization of an Endo-1,4-ß-Xylanase from Wheat Malt.

In this study, an endo-1,4-ß-xylanase was purified from wheat malt following the procedures of ammonium sulfate precipitation, cation-exchange chromatography, and two-step anion-exchange chromatography. The purified endo-1,4-ß-xylanase had a specific activity of 3.94 u/mg, demonstrating a weight average molecular weight (Mw) of approximately 58,000 Da. After LC-MS/MS (Liquid chromatography-tandem mass spectrometry) identification, the purified enzyme had the highest matching degree with a GH10 (Glycoside Hydrolase 10) domain-containing protein from wheat, there were 23 match peptides with a score above the threshold and the prot-cover was 45.5%. The resulting purified enzyme was used to investigate its degradation ability on high viscosity wheat-derived water-extractable arabinoxylan (WEAX). Degradation experiments confirmed that the purified enzyme was a true endo-acting enzyme, which could degrade large WEAX into smaller WEAX. The average degree of polymerization (avDP) and the viscosity of WEAX decreased with the increasing reaction time. The enzyme could degrade a small amount of WEAX into arabinoxylan-oligosaccharides (AXOS) with a degree of polymerization of 2-6, but no monosaccharide was produced. The degradation occurred rapidly in the first 3.5 h and decreased with the further prolongation of reaction time.

[Knockdown of motility-related genes of Komagataeibacter xylinus and its effect on bacterial cellulose synthesis].

Bacterial cellulose (BC) is a biopolymer synthesized by bacteria, which possess excellent characteristics such as high water holding capacity, high crystallinity, and high purity. It is widely used in food, medical, cosmetics, and functional films. Komagataeibacter xylinus is a model strain used in BC synthesis research. In bacteria, motility-related genes are associated with BC synthesis, whereas in Komagataeibacter xylinus CGMCC 2955, the functions of motility-related genes and their effects on BC synthesis are not known. To address this gap, we used the λ Red recombinant system to individually knock out motA, motB, and mot2A respectively, and constructed the knockout strains K. x-ΔmotA, K. x-ΔmotB, and K. x-Δmot2A. Additionally, both motA and motB were disrupted to construct the K. x-ΔmotAB mutant. The results demonstrated that knockout strain K. x-ΔmotAB exhibited the highest BC yield, reaching (5.05±0.26) g/L, which represented an increase of approximately 24% compared to wild-type strains. Furthermore, the BC synthesized by this strain exhibited the lowest porosity, 54.35%, and displayed superior mechanical properties with a Young's modulus of up to 5.21 GPa. As knocking out motA and motB genes in K. xylinus CGMCC 2955 did not reduce BC yield; instead, it promoted BC synthesis. Consequently, this research further deepened our understanding of the relationship between motility and BC synthesis in acetic acid bacteria. The knockouts of motA and motB genes resulted in reduced BC porosity and improved mechanical properties, provides a reference for BC synthesis and membrane structure regulation modification.

Engineering PTS-based glucose metabolism for efficient biosynthesis of bacterial cellulose by Komagataeibacter xylinus.

Bacterial cellulose (BC) is a renewable biomaterial that has attracted significant attention due to its excellent properties and wide applications. Komagataeibacter xylinus CGMCC 2955 is an important BC-producing strain. It primarily produces BC from glucose while simultaneously generating gluconic acid as a by-product, which acidifies the medium and inhibits BC synthesis. To enhance glucose uptake and BC synthesis, we reconstructed the phosphoenolpyruvate-dependent glucose phosphotransferase system (PTSGlc) and strengthened glycolysis by introducing heterologous genes, resulting in a recombinant strain (GX08PTS03; Δgcd::ptsHIcrrE. coli::ptsGE. coli::pfkAE. coli). Strain GX08PTS03 efficiently utilized glucose for BC production without accumulating gluconic acid. Subsequently, the fermentation process was systematically optimized. Under optimal conditions, strain GX08PTS03 produced 7.74 g/L of BC after 6 days of static fermentation, with a BC yield of 0.39 g/g glucose, which were 87.41 % and 77.27 % higher than those of the wild-type strain, respectively. The BC produced by strain GX08PTS03 exhibited a longer fiber diameter along with a lower porosity, significantly higher solid content, crystallinity, tensile strength, and Young's modulus. This study is novel in reporting that the engineered PTSGlc-based glucose metabolism could effectively enhance the production and properties of BC, providing a future outlook for the biopolymer industry.

Enzymatic Properties of endo-1,4-ß-xylanase from Wheat Malt.

BACKGROUND: Arabinoxylan (AX) is the main non-starch polysaccharide in wheat. Wheat malts are traditional raw materials for beer brewing. AX is divided into water-soluble arabinoxylan (WEAX) and waterinsoluble arabinoxylan (WUAX). In the mashing stage of beer production, WUAX in malt is degraded by arabinoxylanase to WEAX, which is further degraded to smaller molecules and retained in the final beer. The viscosity of WEAX is related to its molecular weight. WEAX with higher molecular weight and viscosity can increase viscosity and turbidity and reduce filtration speed of wort and beer; WEAX with moderate molecular weight and viscosity contributes to the foaming characteristics and foam stability, and promotes the taste and texture of a beer; WEAX with small molecular weight has the functions of anti-tumor and lowering blood pressure and is regarded as a prebiotic. Because WEAXs with different molecular weight and properties have different impacts on the beer brewing process and qualities of the final beer, it becomes more important to control the degradation of AX during the brewing process of a beer. Endo-1,4-ß-xylanase (EC 3.2.1.8) is the most important AX degrading enzyme, which cleaves the ß -xylosidic bond between two d-xylopyranosyl residues linked in ß-(1,4). The study of enzymatic properties of endo-1,4-ß-xylanase from wheat malt is very important for the rational formulation of the content and molecular weight of WEAX in wort and beer during the mashing procedure when using wheat malt as the main raw materials. OBJECTIVE: In this article, our motivation is to study the enzymatic properties (including optimum pH and temperature, pH and temperature stability, the effect of inhibitors) of wheat malt endo-1,4-ß-xylanase. METHODS: In this article, we prepared crude enzyme according to the method of Guo with minor modifications. The endo-1,4-ß-xylanase activity was determined according to the method of Biely in the previous report with minor modifications. The 0.5 mL crude enzyme sample was mixed with 0.5 mL 1 mg/mL 4-O-methyl-dglucurono- d-xylan dyed with Remazol Brilliant Blue R (RBBR-Xylan) solution, intensively mixed, and incubated at 40 °C for exactly 90 min. The reaction was stopped by precipitation using 2 mL absolute ethanol, and the reaction mixture was stirred acutely and placed at room temperature for 30 min. Then, the mixture was mixed again and centrifuged at 6000 g for 10 min. The supernatant was collected and the absorbance was measured at 590 nm. Absolute ethanol and RBBR-Xylan were added to the control tubes first, and after the reaction was completed, the crude enzyme sample was added. One unit of endo-1,4-ß-xylanase was defined as at pH 5.5 and 40 °C liberate 1 µmol xylose equivalents in 1 min per g dry wheat malt. RESULTS: The results showed that the optimal activity of endo-1,4-ß-xylanase was achieved at pH 5.5-6.0, and the enzyme was extremely stable at pH 4.5, 5.5 and 6.5 after incubation for 30, 50 and 60 min, respectively. The optimal temperature was 40-45 °C and the deactivation temperature was 75 °C. Endo-1,4-ß-xylanase was stable at 20 °C and 40 °C; the stability was slightly decreased at 50 °C and rapidly decreased at 55 °C. The enzyme activity was mildly inhibited by K+, Na+, and Pb2+, moderately inhibited by Ca2+, Mg2+ and Mn2+ and severely inhibited by Cu2+, Ag+ and EDTA. CONCLUSION: We have got the enzymatic properties of endo-1,4-ß-xylanase from wheat malt, so during wort mashing, we could apply this research result to carry out the rational formulation of the content and molecular weight of WEAX in wort and beer during the mashing procedure when using wheat malt as the main raw materials. Expected to solve the technical problems such as high viscosity, slow filtration speed and so on, but also highlight the typical flavors of WEAX such as rich and persistent foam and mellow texture during the brewing process of a beer.