Enzyme and Microbial Technology最新文献

The sensing modified electrode was prepared using glucose oxidase immobilized onto vanadium pentoxide xerogel with glass/FTO as support electrode to evaluate the possibility to construct a V₂O₅/GOx Extended Gate Field Effect Transistor biosensor. Previously, our studies exhibited a sensitivity of V₂O₅ of 58.1 mV/pH. The use of Nafion® onto V₂O₅/GOx caused a decrease of mass loss after several cycles compared to the modified electrode without Nafion® during the EQCM and cyclic voltammetrics studies. Electrical characterization of V₂O₅/GOx demonstrated a tendency to stability after 200 s as a function of applied current. In presence of glucose and in different pH, the current decreased when the glucose concentration increased due to the lower active sites of enzyme. After ten voltammetric cycles, the total charge tends to structural stability. In pH = 5.0, the modified electrode based on V₂O₅/GOx Extended Gate Field Effect Transistor presented more tendency to sensitivity in different concentration of glucose.

Poly(ethylene furanoate) (PEF) plastic is a 100% renewable polyester that is currently being pursued for commercialization as the next-generation bio-based plastic. This is in line with growing demand for circular bioeconomy and new plastics economy that is aimed at minimizing plastic waste mismanagement and lowering carbon footprint of plastics. However, the current catalytic route for the synthesis of PEF is impeded with technical challenges including high cost of pretreatment and catalyst refurbishment. On the other hand, the semi-biosynthetic route of PEF plastic production is of increased biotechnological interest. In particular, the PEF monomers (Furan dicarboxylic acid and ethylene glycol) can be synthesized via microbial-based biorefinery and purified for subsequent catalyst-mediated polycondensation into PEF. Several bioengineering and bioprocessing issues such as efficient substrate utilization and pathway optimization need to be addressed prior to establishing industrial-scale production of the monomers. This review highlights current advances in semi-biosynthetic production of PEF monomers using consolidated waste biorefinery strategies, with an emphasis on the employment of omics-driven systems biology approaches in enzyme discovery and pathway construction. The roles of microbial protein transporters will be discussed, especially in terms of improving substrate uptake and utilization from lignocellulosic biomass, as well as from depolymerized plastic waste as potential bio-feedstock. The employment of artificial bioengineered microbial consortia will also be highlighted to provide streamlined systems and synthetic biology strategies for bio-based PEF monomer production using both plant biomass and plastic-derived substrates, which are important for circular and new plastics economy advances.

Eukaryotic sialyltransferases play key roles in many physiological and pathological events. The expression of active human recombinant sialyltransferases in bacteria is still challenging. In the current study, the genes encoding human N-acetylgalactosaminide α2,6-sialyltransferase V (hST6GalNAc V) and N-acetylgalactosaminide α2,6-sialyltransferase VI (hST6GalNAc VI) lacking the N-terminal transmembrane domains were cloned into the expression vectors, pET-32a and pET-22b, respectively. Soluble and active forms of recombinant hST6GalNAc V and hST6GalNAc VI when coexpressed with the chaperone plasmid pGro7 were successfully achieved in Escherichia coli. Further, lactose (Lac), Lacto-N-triose II (LNT II), lacto-N-tetraose (LNT), and sialyllacto-N-tetraose a (LSTa) were used as acceptor substrates to investigate their activities and substrate specificities. Unexpectedly, both can transfer sialic acid onto all those substrates. Compared with hST6GalNAc V expressed in the mammalian cells, the recombinant two α2,6-sialyltransferases in bacteria displayed flexible substrate specificities and lower enzymatic efficiency. In addition, an important human milk oligosaccharide disialyllacto-N-tetraose (DSLNT) can be synthesized by both human α2,6-sialyltransferases expressed in E. coli using LSTa as an acceptor substrate. To the best of our knowledge, these two active human α2,6-sialyltransferases enzymes were expressed in bacteria for the first time. They showed a high potential to be applied in biotechnology and investigating the molecular mechanisms of biological and pathological interactions related to sialylated glycoconjugates.

Cytochrome P450s (CYPs) regulate plant growth and stress responses by producing diverse primary and secondary metabolites. However, the function of many plant CYPs remains unknown because, despite their structural similarity, predicting the enzymatic activity of CYPs is difficult. In this study, one member of the CYP736A subfamily (CYP736A61) from tomatoes was isolated and characterized its enzymatic functions. CYP736A61 was successfully expressed in Escherichia coli through co-expression with molecular chaperones. The purified CYP736A61 showed hydroxylation activity toward 7-ethoxycoumarin, producing 7-hydroxycoumarin or 3-hydroxy 7-ethoxycoumarin. Further substrate screening revealed that dihydrochalcone and stilbene derivates (resveratrol and polydatin) are the substrates of CYP736A61. CYP736A61 also mediated the hydroxylation of resveratrol and polydatin, albeit with low activity. Importantly, CYP736A61 mediated the cleavage of resveratrol and polydatin as well as pinostilbene and pterostilbene. Interestingly, CY736A61 also converted phloretin to naringenin chalcone. These results suggest that CYP736A61 is a novel CYP enzyme with stilbene cleavage activity.

In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6 U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ˚C) with a 2.83 mg/mL and 0.063 μmol/minute for K_m and V_max, respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43 mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7 mg/mL, the time for the fabric to absorb water was 19.77 seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38 seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring.

Phenylalanine ammonia-lyase (PAL) plays a pivotal role in the biosynthesis of phenylalanine. PAL from Zea mays (ZmPAL2) exhibits a bi-function of direct deamination of L-phenylalanine (L-Phe) or L-tyrosine(-L-Tyr) to form trans-cinnamic acid or p-coumaric acid. trans-Cinnamic acid and p-coumaric acid are mainly used in flavors and fragrances, food additives, pharmaceutical and other fields. Here, the Activity of ZmPAL2 toward L-Phe or L-Tyr was improved by using semi-rational and rational designs. The catalytic efficiency (k_cat/K_m) of mutant PT10 (V258I/I459V/Q484N) against L-Phe was 30.8 μM⁻¹ s⁻¹, a 4.5-fold increase compared to the parent, and the catalytic efficiency of mutant PA1 (F135H/I459L) to L-tyrosine exhibited 8.6 μM⁻¹ s⁻¹, which was 1.6-fold of the parent. The yield of trans-cinnamic acid in PT10 reached 30.75 g/L with a conversion rate of 98%. Meanwhile, PA1 converted L-Tyr to yield 3.12 g/L of p-coumaric acid with a conversion rate of 95%. Suggesting these two engineered ZmPAL2 to be valuable biocatalysts for the synthesis of trans-cinnamic acid and p-coumaric acid. In addition, MD simulations revealed that the underlying mechanisms of the increased catalytic efficiency of both mutant PT10 and PA1 are attributed to the substrate remaining stable within the pocket and closer to the catalytically active site. This also provides a new perspective on engineered PAL.

The utilisation of carbonic anhydrase (CA) in CO₂ sequestration is becoming prominent as an efficient, environment friendly and rapid catalyst for capturing CO₂ from industrial emissions. However, the application of CA enzyme in soluble form is constrained due to its poor stability in operational conditions of CO₂ capture and also production cost of the enzyme. Addressing these limitations, the present study focuses on the surface display of CA from Bacillus halodurans (BhCA) on E coli aiming to contribute to the cost-effectiveness of carbon capture through CA technology. This involved the fusion of the BhCA-encoding gene with the adhesion molecule involved in diffuse adherence (AIDA-I) autotransporter, resulting in the efficient display of BhCA (595 ± 60 U/gram dry cell weight). Verification of the surface display of BhCA was accomplished by conjugating with FITC labelled anti-his antibody followed by fluorescence-activated cell sorting (FACS) and cellular fractionation in conjunction with zymography. Biochemical characterisation of whole-cell biocatalyst revealed a noteworthy enhancement in thermostability, improvement in the thermostability with T_1/2 of 90 ± 1.52 minutes at 50 ˚C, 36 ± 2.51 minutes at 60 ˚C and18 ± 1.52 minutes at 80˚C. Surface displayed BhCA displayed remarkable reusability retaining 100% activity even after 15 cycles. Surface displayed BhCA displayed highly alkali stable nature like free counterpart in solution. The alkali stability of the surface-displayed BhCA was comparable to its free counterpart in solution. Furthermore, the study investigated the impact of different metal ions, modulators, and detergents on the whole-cell biocatalysts. The present work represents the first report on surface display of CA utilising the AIDA-1 autotransporter.

Isoquercetin and D-allulose have diverse applications and significant value in antioxidant, antibacterial, antiviral, and lipid metabolism. Isoquercetin can be synthesized from quercetin, while D-allulose is converted from D-fructose. However, their production scale and overall quality are relatively low, leading to high production costs. In this study, we have devised a cost-effective one-pot method for biosynthesizing isoquercetin and D-allulose using a whole-cell biocatalyst derived from quercetin and sucrose. To achieve this, the optimized isoquercetin synthase and D-allulose-3-epimerase were initially identified through isofunctional gene screening. In order to reduce the cost of uridine diphosphate glucose (UDPG) during isoquercetin synthesis and ensure a continuous supply of UDPG, sucrose synthase is introduced to enable the self-circulation of UDPG. At the same time, the inclusion of sucrose permease was utilized to successfully facilitate the catalytic production of D-allulose in whole cells. Finally, the recombinant strain BL21/UGT-SUS+DAE-SUP, which overexpresses MiF3GT^MUT, GmSUS, EcSUP, and DAEase, was obtained. This strain co-produced 41±2.4 mg/L of isoquercetin and 5.7±0.8 g/L of D-allulose using 120 mg/L of quercetin and 20 g/L of sucrose as substrates for 5 h after optimization. This is the first green synthesis method that can simultaneously produce flavonoid compounds and rare sugars. These findings provide valuable insights and potential for future industrial production, as well as practical applications in factories.

A strain high-performance of esterase producing bacteria was screened from soil, which could selectively hydrolyze D-homoserine lactone from its racemate to achieve the resolution of L- homoserine lactone with more than 99% e.e. in 48% yield. L-homoserine lactone building block was then converted to L-α-amino-γ-bromobutyronic acid chiral blocks, which reacted with various nucleophilic reagent modules could to be applied to prepare L-γ- substituted α-amino acids such as L-selenomethionine, L-methionine, L-glufosinate and L-selenocystine. Its advantages included high selectivity of biocatalytic resolution reactions, high optical purity of products, racemic recycle of D-substrates and modular reaction, which simplified the production process of these products and highlighted the power of biological manufacturing.

Prunin of desirable bioactivity and bioavailability can be transformed from plant-derived naringin by the key enzyme α-L-rhamnosidase. However, the production was limited by unsatisfactory properties of α-L-rhamnosidase such as thermostability and organic solvent tolerance. In this study, biochemical characteristics, and hydrolysis capacity of a novel α-L-rhamnosidase from Spirochaeta thermophila (St-Rha) were investigated, which was the first characterized α-L-rhamnosidase for Spirochaeta genus. St-Rha showed a higher substrate specificity towards naringin and exhibited excellent thermostability and methanol tolerance. The K_m of St-Rha in the methanol cosolvent system was decreased 7.2-fold comparing that in the aqueous phase system, while k_cat/K_m value of St-Rha was enhanced 9.3-fold. Meanwhile, a preliminary conformational study was implemented through comparative molecular dynamics simulation analysis to explore the mechanism underlying the methanol tolerance of St-Rha for the first time. Furthermore, the catalytic ability of St-Rha for prunin preparation in the 20% methanol cosolvent system was explored, and 200 g/L naringin was transformed into 125.5 g/L prunin for 24 h reaction with a corresponding space-time yield of 5.2 g/L/h. These results indicated that St-Rha was a novel α-L-rhamnosidase suitable for hydrolyzing naringin in the methanol cosolvent system and provided a better alternative for improving the efficient production yield of prunin.