查看更多>>摘要：The gamma-cyclodextrin glycosyltransferase (gamma-CGTase) from Evansella caseinilytica was expressed on the cell surface of E. coli using pAIDA-I autotransporter and was further utilized in the conversion of starch to gamma-cyclodextrins (CDs). Maximum cyclization activity of 2.28 +/- 0.46 U/g biomass was achieved after 3 h of induction using 0.1 mM IPTG at 37 degrees C. Surface expression of gamma-CGTase was confirmed using flow cytometry employing a FITC-conjugated anti-HIS antibody. Biochemical characterization of surface-displayed gamma-CGTase revealed optima at pH 10.0 and 40 degrees C along with a t(1/2) of 24.75 mM at 50 degrees C. The K-m and V-max values on soluble potato starch were 10.94 mg/ml and 4.33 mu moles min(-1) g(-1) DCW respectively, and the activation energy was calculated to be 89.8 kJ/mol. The surface displayed gamma-CGTase was further utilized for CD production and specifically, gamma-CD conversion was obtained. The maximum conversion was achieved at 50 degrees C, pH 9.0 using soluble potato starch (2.5%; w/v) taking a final enzyme concentration of 0.6 U/g starch. The surface-displayed gamma-CGTase was able to convert soluble potato starch (2.5%) into gamma-CDs with a 72.7% specific yield and no other peaks corresponding to alpha- and beta-CDs were observed on HPLC. The enzyme was found to be similar to 100% operationally stable for up to 2 consecutive cycles of 24 h, with > 75% storage stability at - 20 degrees C even after 7 days.
Lee, Ji YeonKim, HayeongMoon, YedamKwak, Sohyung...
8页
查看更多>>摘要：This study aimed to enhance the water solubility and antioxidant properties of mangiferin by transglucosylation using cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. The highest mangiferin to mangiferin glucoside conversion yield achieved was 88.9% using 60 mU/mL CGTase, 25 mM mangiferin, and 10% starch (w/v), with incubation at 60 ?C for 10 h. The product of transglucosylation was purified and its chemical structure was determined to be glucosyl-alpha-(1 -> 4)-mangiferin (MGF-g1) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. The water solubility of MGF-g1 was 5,093 times higher than that of mangiferin. MGF-g1 exhibited 1.6-fold higher 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, 1.24-fold higher oxygen radical antioxidant capacity, and 1.19-fold higher ferric reducing ability power, compared to mangiferin. Moreover, the cyclooxygenase-2 inhibitory activity (IC50) of mangiferin and MGF-g1 were 76.44 +/- 11.7 mu M and 59.74 +/- 2.8 mu M, respectively. Our results suggest that the novel MGF-g1 has potential applications as a functional material in the food and pharmaceutical industries.
查看更多>>摘要：Phosphatases catalyze the irreversible dephosphorylation of phosphate-containing compounds, and hence can be applied as the final enzymatic step for the synthesis of various biochemicals. However, the extensive substrate spectrums of phosphatases impose a great challenge for efficient biomanufacturing. Characterization of phosphatases is therefore of extreme importance. In this study, MmPase, a putative HAD phosphatase from Methanothermobacter marburgensis, was expressed, purified, and characterized. Recombinant MmPase was readily expressed in Escherichia coli, and required metal ions such as Mn2+ or Mg2+ to function. MmPase worked optimally at 50 degrees C, pH 6.5, and exhibited a half-life of 6.5 h under this condition. Among all substrates tested, MmPase established the highest dephosphorylation activity against D-tagatose 6-phosphate, and was relatively specific for this substrate than for D-glucose 1-phosphate, D-glucose 6-phosphate, and D-fructose 6-phosphate. Therefore, MmPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D-tagatose from maltodextrin, and achieved a product yield of 37.6%. Our studies of MmPase provided a promising strategy for the economic and efficient production of D-tagatose in the future.
查看更多>>摘要：The apparent solubility and bioavailability of hydrophobic compounds, the main problems in the bioremediation process, can be overcome by bacteria producing biosurfactants. Lipopeptide biosurfactants, as high-efficiency biosurfactants with biological activity, can be synthesized by nonribosomal peptide synthetase (NRPS). In this study, two strains, Paenibacillus lautus A (HC_A) and Lysinibacillus fusiformis B (HC_B), which can efficiently produce lipopeptide biosurfactants, were screened. The NRPS gene sequences of these two strains were obtained by whole-genome sequencing. The module of synthetic surfactant NRPS from HC_A and the module of synthetic surfactants NRPS and sfp from HC_B were successfully cloned into plasmids and then expressed in E. coli (namely, E. coli pHC_A-NRPS and E. coli pHC_B-NRPS-sfp, respectively). Finally, stable lipopeptide biosurfactants could be expressed heterologously. Our research shows that the constructed recombinant bacteria E. coli pHC_A-NRPS and E. coli pHC_B-NRPS-sfp can heterologously express the NRPS module (graphical abstract is shown in Fig 1). Remarkably, the lipopeptide biosurfactants produced by the constructed recombinant bacteria of the NRPS gene have good emulsifying activity on tributyrin. Meanwhile, adding SiO2 nanoparticles and surface-modified carbon black can synergistically enhance the emulsifying effect, which has not been reported before. This research may deepen the comprehension of microbial strains that produce surfactants to emulsify environmental pollutants and perform bioremediation.
查看更多>>摘要：Chitinases are present in diverse form of organisms from bacteria, fungi, insects, plants. Plant chitinases are part of pathogenesis-related proteins. When plant (host) cells are under pathogen stress, plant chitinases are strongly expressed and hence plant chitinases play a critical part against fungal pathogens. Chitinases are also found to be involved in various abiotic stress responses like wounding, osmotic pressure, cold, heavy metal stress, salt in plants. Understanding of the plant chitinases will provide an insight for improving the pathogenic activity of various potential biocontrol strains and to develop novel pathogen resistant strategies for exploring their roles with regards to plant defense. The present review covers the detailed account of potential and relevance of plant chitinases for controlling pathogens infection in plant and prospecting to improve plant defense, growth and yield.
查看更多>>摘要：In the bioproduction of glutaric acid, an emerging bioplastic monomer, alpha-ketoglutaric acid (alpha-KG) is required as an amine acceptor for 4-aminobutyrate aminotransferase (GabT)-driven conversion of 5-aminovalerate (5-AVA) to glutarate semialdehyde. Herein, instead of using expensive alpha-KG, an indirect alpha-KG supply system was developed using a relatively cheap alternative, monosodium glutamate (MSG), for L-glutamate oxidase (Gox)based whole-cell conversion. Using 200 mM 5-AVA and 30 mM MSG initially with Gox, 67.1 mM of glutaric acid was produced. By applying the stepwise feeding strategy of MSG, the glutaric acid production capability was increased to 159.1 mM glutaric acid with a conversion yield of 79.6%. In addition, a buffer-free one-pot reaction from L-lysine was also applied in a 5 L bioreactor to evaluate its industrial applicability, resulting in a conversion yield of 54.2%. The system developed herein might have great potential for the large-scale, economically feasible production of glutaric acid by whole-cell conversion.
查看更多>>摘要：D-Allulose 3-epimerase (DAE) is promising to be used for the production of the rare sugar D-allulose in industry. However, the poor thermostability and low catalytic efficiency limited its large-scale industrial applications. A dual-enzyme screening method was developed to measure the activity of the D-allulose 3-epimerase from Clostridium cellulolyticum H10 by employing a xylose isomerase, enabling high-throughput screening of mutants with higher thermostability. After two rounds of directed evolution, the H56R, Q277R, H56R/Q277R and H56R/ Q277R/S293R variants were obtained with 1.9, 1.8, 3.5 and 7.1 ? improvement in T-50(5) , the temperature at which the enzyme activity becomes half of the original after the 5 min treatment and 3.1-, 4.2-, 4.4-and 9.47-fold improvement in the half life at 60 ?, respectively, compared with the wild-type enzyme. Among them, triple mutant H56R/Q277R/S293R showed significant improvement in k(cat)/K-m compared to the wild type enzyme. Molecular dynamics simulations provided the insights into improving the thermostability by three arginine mutations. The research will aid the development of industrial biocatalysts for the production of D-allulose.
Jiang, BoDai, YiweiChen, JingjingHassanin, Hinawi A. M....
8页
查看更多>>摘要：Maltoheptaose (G7) is one of the mixtures of maltodextrin widely used in the food, pharmaceutical, and cosmetics industries. A genetically engineered strain, which simultaneously expressed cyclodextrin glucanotransferase (CGTase) from Gracilibacillus alcaliphilus SK51.001 and cyclomaltodextrinase (CDase) from Bacillus sphaericus E-244, two enzymes, was constructed by cloning the above two genes into a plasmid and transformed into the host Escherichia coli BL21(DE3) (E. coli) strain, resulted in recombinant cells harboring the vector pETDuet-GaCGT/BsCD (pGaBs). These cells were used as whole-cell catalysts for the biotransformation of G7 from the inexpensive substrate (starch). Due to the high molecular weight of starch, the cell membrane prevents the entry of starch into the cellular system. Therefore, the pGaBs cell wall was permeabilized by lysozyme, EDTA, and heat treatment. After reaching the optimized conditions of permeabilized pGaBs cell amount, lysozyme amount, reaction temperature, and metal ion concentration, approximately 4.1 g/L of G7 was produced from 30 g/L starch in 1 h with the addition of Ca2+. This co-expression system offers a one-pot synthesis approach to the production of G7 using an inexpensive substrate, avoiding enzyme purification steps.
查看更多>>摘要：Yeasts represent a group of the microorganisms most frequently seen in biotechnology. Recently, the class 2 type II CRISPR system (CRISPR/Cas9) has become the principal toolbox for genome editing. By efficiently implementing genetic manipulations such as gene integration/knockout, base editor, and transcription regulation, the development of biotechnological applications in yeasts has been extensively promoted. The genome-level tools based on CRISPR/Cas9, used for screening and identifying functional genes/gene clusters, are also advancing. In general, CRISPR/Cas9-assisted editing tools have gradually become standardized and function as host-orthogonal genetic systems, which results in time-saving for strain engineering and biotechnological application processes. In this review, we summarize the key points of the basic elements in the CRISPR/Cas9 system, including Cas9 variants, guide RNA, donors, and effectors. With a focus on yeast, we have also introduced the development of various CRISPR/Cas9 systems and discussed their future possibilities.
NSTL
Elsevier