High Enzyme Activity Research Articles

Protein Engineering of Nicotinamide Riboside Kinase Based on a Combinatorial Semirational Design Strategy for Efficient Biocatalytic Synthesis of Nicotinamide Mononucleotides.

Industrial biosynthesis of β-nicotinamide mononucleotide (β-NMN) lacks a highly active nicotinamide riboside kinase for the phosphorylation process. Cumbersome preprocessing steps and excessive ATP addition contribute to its increased cost. To tackle these challenges, a docking combination simulation (DCS) semirational mutagenesis strategy was designed in this study, combining various modification strategies to obtain a mutant NRK-TRA with 2.9-fold higher enzyme activity. Molecular dynamics simulations and structural analysis demonstrate the enhancement of its structural stability. High-density fermentation was achieved through a 5 L fermentation tank, with a titer reaching 208.3 U/mL, the highest in the current report. An ATP-cycling whole-cell catalytic system was employed and optimized by introducing a polyphosphate kinase 2 (PPK2) recombinant strain, and 15.16 g/L β-NMN was obtained through a series of batch transformation experiments. This study provides a new strategy for the efficient screening of highly active enzyme variants and offers a green and promising biotransformation system for NMN production.

Direct Preparation of Alginate Oligosaccharides from Brown Algae by an Algae-Decomposing Alginate Lyase AlyP18 from the Marine Bacterium Pseudoalteromonas agarivorans A3

Alginate oligosaccharides (AOs), derived from alginate degradation, exhibit diverse biological activities and hold significant promise in various fields. The enzymatic preparation of AOs relies on alginate lyases, which offers distinct advantages. In contrast to the conventional use of sodium alginate derived from brown algae as the substrate for the enzymatic preparation of AOs, AO preparation directly from brown algae is more appealing due to its time and energy efficiency. Thus, the identification of potent alginate lyases and cost-effective brown algae substrates is crucial for optimizing AO production. Herein, we identified and characterized an alginate lyase, AlyP18, capable of efficiently decomposing algae, from a marine bacterium Pseudoalteromonas agarivorans A3 based on secretome analysis. AlyP18 is a mesothermal, endo-type and bifunctional alginate lyase with high enzymatic activity. Two brown algae substrates, Laminaria japonica roots and Macrocystis pyrifera, were used for the AO preparation by AlyP18. Upon optimization of AlyP18 hydrolysis parameters, the substrate degradation efficiency and AO production reached 53% and ~32% for L. japonica roots, respectively, and 77% and ~46.5% for M. pyrifera. The generated AOs primarily consisted of dimers to pentamers, with trimers and tetramers being dominant. This study provides an efficient alginate lyase and alternative brown algal feedstock for the bioconversion of high-value AOs from brown algae.

Degradation of High Concentrations of Anthracene Using White-Rot Wood-Inhabiting Fungi and Investigation of Enzyme Activities

Owing to the production of lignin-modifying enzymes (LMEs), white-rot fungi (WRF) such as polypores are potent organisms in the biodegradation of xenobiotic pollutants. The nonspecific function of LMEs including laccase and manganese peroxidase (MnP), has enabled the use of WRF in biotechnological applications, particularly in bioremediation. In this study, 12 strains from nine white-rot basidiomycete genera viz., Ganoderma, Inocutis, Irpex, Lentinus, Lenzites, Oxyporus, Peniophora, Sanghuangporus, and Trametes were isolated from Iran and identified using morphological and molecular tools. The enzyme activity of laccase and manganese peroxidase that directly correlated with the biodegradation were determined, and the strains with the highest enzyme activities were evaluated for their ability to degrade 400 mg/L of anthracene over 28 days. Gas chromatography with flame ionization detector (GC-FID) revealed that four polypores viz., Trametes versicolor v21te, T. versicolor v22da, T. hirsuta, and Oxyporus sp. degraded 64%, 52%, 34%, and 20% of the anthracene, respectively. According to our analysis, the examined indigenous WRF are potentially useful candidates for the development of new mycoremediation techniques to degrade polycyclic aromatic hydrocarbons (PAHs).

Exploring the potential of triticale lines for bioethanol production

Aim: Triticale is a well adaptable crop, tolerant of disease and abiotic stresses, and able to grow with good yields even in poor soil, thus representing a good choice to develop a new industrial agri-chain in Italy in a sustainability contest, to cope with its soil problems due to incoming desertification. Methods: Two triticale elite lines were grown in marginal lands in controlled field experiments. The lines were harvested at two different development stages, namely green mass and seeds, and suitable standard protocols were applied to test their potential to produce bioethanol in line with the emerging bioenergy processes. Results: The protocols applied were able to obtain bioethanol with a good yield from both feedstocks. In particular, very efficient fermentation kinetics was observed using seed feedstock, with a sharp curve between 15 h and 24 h, reaching 84% of the total alcohol obtained (final time 72 h). Conclusions: Therefore, the results of this research point to new sustainable potential for industrial applications of triticale crops in Italy. Furthermore, the high activity of the endogenous amylolytic enzymes, mainly α-amylase, and the high starch content suggest the potential use of triticale in other industrial applications, like the brewing industry.

Long term impact of manuring, fertilization and liming on soil resilience, enzyme activities and productivity under maize-wheat system in an acidic Alfisol

Aim: This study was carried out to evaluate the long-term impact of manuring, fertilization and liming on soil resilience, enzyme activities and system productivity under changing climate scenarios. Methodology: The soil samples were collected from 67 year old long-term experiment going on, at Birsa Agricultural University, Jharkhand during 2022-23 under maize-wheat cropping system from seven treatments, viz. T1: control, T2: 100 % N, T3: 100 % FYM, T4: ½ (N+FYM) + PX/2 + KY/2, T5: 100 % NPK, T6: Lime + NPK, T7: Lime + FYM +P(A-X) + K(B-Y). To assess soil resistance and resilience, surface samples were subjected to with and without heat stress (48±2°C for 80 hrs) and substrate addition (0.02 g glucose g-1 soil). Results: Application of FYM/lime with NPK significantly improved bulk density, pH, SOC and system productivity. The highest enzyme activities and cumulative respiration were observed in lime + FYM + P(A-X) + K(B-Y) treatment. It exhibited the highest resistance index (0.92) and resilience index (0.34). Interpretation: The integrated use of inorganic, organic along with liming proved to be a useful management strategy for maintaining soil quality, resistance and resilience, and crop productivity in acid soil of Jharkhand. Key words: Enzyme, Heat stress, Maize-wheat system, Soil acidification, Substrate induced respiration, Wheat-maize

Isolation and identification of cellulose decomposition bacteria from the soil of Yunnan province, China

In this experiment sodium carboxymethyl cellulose (CMC-Na) was used as the only carbon source to isolate cellulose decomposing bacteria from the soil of Qiongzhusi Forest Park in Kunming, Yunnan Province. The ratio of hydrolytic circle diameter and colony diameter formed by the strain on cellulose Congo red medium was determined for primary screening, and then the filter paper disintegration test and CMC enzyme activity assay were used for rescreen. A cellulose decomposing bacterium (HS-2) with high enzyme activity was obtained, and its enzyme activity was up to 16.42 U/ml. (U means the unit of activity of enzymes, activity, unit) Gram stain and Spore stain reaction showed that HS-2 strain was Gram-positive Bacillus. The Bacillus strain was identified as Bacillus pumilus on the basis of physiological and biochemical tests, and 16S rDNA sequence analysis. Bangladesh J. Bot. 52(3): 635-641, 2024 (September) Special

Identification and Growth Characteristics of Cellulase Producing Bacteria

This study was envisaged to from fresh cow dung using the Congo red staining method, and screened by measuring their endoglucanase activity (CMC enzyme activity), exoglucanase activity (Cex enzyme activity), and filter paper enzyme activity (FPA enzyme activity). Subsequently, the molecular, morphological characteristics, and growth characteristics were studied. Three strains showed clear transparent circles on the Congo red cellulose medium. LY3 exhibited strong enzyme production ability, with relatively higher enzyme activity than the other two strains. LY3 was found to be a dominant strain that required a temperature of 40℃and pH 7.0 for optimal growth. Based on the molecular and morphological characteristics, the three strains were preliminarily identified as Bacillus. Bangladesh J. Bot. 53(3): 849-856, 2024 (September) Special

Mammalian D-Cysteine controls insulin secretion in the pancreas

D-amino acids are being recognized as important molecules in mammals with function. This is a first identification of endogenous D-cysteine in mammalian pancreas. D-cysteine is synthesized by serine racemase (SR) and SR−/− mice produce 6–10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (Streptozotocin (STZ) and Non Obese Diabetes (NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR−/− mice show decreased cAMP in the pancreas, lower DNA methyltransferase enzymatic and promoter activities followed by reduced phosphorylation of CREB (S133), resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR−/− mice. Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion.

Periodic Paralysis in a Child With Thermosensitive Mitochondrial Trifunctional Protein Deficiency.

Mitochondrial trifunctional protein (MTP) deficiency is a fatty acid oxidation disorder associated with a spectrum of phenotypes. Patients with high residual enzyme activity tend to have milder phenotypes, and recently, fever-induced episodic myopathy was reported in association with a thermosensitive form of MTP deficiency. We report a 10-year-old male with recurrent episodes of acute flaccid paralysis involving upper and lower extremities in association with bulbar muscle weakness in the context of febrile illness, a phenotype reminiscent of recurrent periodic paralysis. The episodes started at the age of 3 years and have always been followed by full recovery within 1-2 weeks with no residual weakness. Whole exome sequencing revealed a homozygous c.2132C > T, p.(Pro711Leu) variant in HADHA. The variant leads to mildly reduced long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) and long-chain ketoacyl-CoA thiolase (LCKAT) enzyme activities and reduced MTP protein expression in patient's fibroblasts when cultured at 37°C. Enzyme activities and MTP protein expression diminished when fibroblasts were cultured at 40°C. This is the first published report of confirmed recurrent periodic paralysis as a manifestation of a thermosensitive form of MTP deficiency, and it calls for this condition to be considered when evaluating patients with recurrent periodic paralysis given therapeutic implications.

Thiopurine S-methyl Transferase (TPMT) Enzyme Level in Healthy Sudanese Population

Background: Thiopurine drugs have limited use due to their toxicity, related to the enzyme thiopurine S-methyl transferase (TPMT) activity, which varies between individuals. This is the first study in Sudan, which aimed to assess the TPMT phenotypic status of healthy Sudanese volunteers. Methods: A total of 177 healthy volunteers from Sudan were included in the study. TPMT enzymatic activities were measured using the ELISA serum protocol. We used SPSS to analyze the data and determined enzyme level categories and normal range with Z scores and quartile tests. The Sudan Medical Specialization Board (SMSB) Ethical Committee approved the study. Results: There were 117 males and 60 females among the volunteers, with ages ranging from 16 to 70 years and a mean age ± SD of 28.0 ±1 0.2, median = 24. Most candidates were from the Afro-Asiatic linguistic group (64.5%), followed by Nilo-Saharan (18.6%) and Niger-Kordofanian (16.9%). The TPMT enzyme level ranged between 0.17 and 9.5 ng/ml, with a mean of 2.26 ± 0.75 ng/ml. The quartile classification included very low enzyme (<0.76 ng/ml) seen in 4 candidates (2.3%), intermediate low (0.76-1.4 ng/ml) seen in 34 (19.2%), the normal range (1.5 – 3.75 ng/ml) seen in 119 (67.2%), and high enzyme activity (>3.76 ng/ml) seen in 20 (11.3%). No significant correlations between age, sex, and ethnic groups were recorded. Conclusion: The normal TPMT enzyme activity is between 1.5 and 3.76 ng/ml. A higher prevalence of TPMT deficiency was recorded and compared with international studies. Pretreatment screening using serum ELISA test for TPMT enzyme activity should be used to predict the risk of toxicity.

Earlier endosomal escape improves the catalytic activity of delivered enzyme cargo.

AbstractThere is enormous interest in strategies to efficiently traffic biologics–proteins, nucleic acids, and complexes thereof–into the mammalian cell cytosol and internal organelles. Not only must these materials reach the appropriate cellular locale fully intact and in therapeutically relevant concentrations, they must also retain activity upon arrival. The question of residual activity is especially critical when delivery involves exposure to the late endocytic pathway, whose acidic lumenal environment can denature and/or degrade internalized material. ZF5.3 is a compact, stable, rationally designed mini-protein that efficiently escapes intact from late endocytic vesicles, with or without covalently linked protein cargo. Here, using insights from mechanistic studies on the pathway of endosomal escape and classic knowledge regarding the bioinorganic chemistry of zinc(II) coordination in small proteins, we re-designed the sequence of ZF5.3 to successfully alter the timing (but not the efficiency) of endosomal escape. The new mini-protein we describe, AV5.3, escapes earlier than ZF5.3 along the endocytic pathway with no loss in efficiency, with or without enzyme cargo. More importantly, earlier endosomal escape translates into higher enzymatic activity upon arrival in the cytosol. Delivery of the pH-sensitive protein dihydrofolate reductase (DHFR) with AV5.3 results in substantial catalytic activity in the cytosol, whereas delivery with ZF5.3 does not. The activity of AV5.3-DHFR upon delivery is sufficient to rescue a genetic DHFR deletion in CHO cells. This work provides evidence that programmed trafficking through the endosomal pathway is a viable strategy for the efficient cytosolic delivery of therapeutic proteins.Abstract Figure

Microbial inoculation accelerates rice straw decomposition by reshaping structure and function of lignocellulose-degrading microbial consortia in paddy fields

Inoculating lignocellulose-degrading microorganisms can accelerate straw decomposition in paddy field; however, the relationship between indigenous and inoculated microorganisms remains unclear. This study explored the effects of microbial inoculation on straw decomposition, microbial community, lignocellulose-degrading consortia, and associated functional genes. After inoculation, straw degradation rate increased by up to 4.9 %, and the rice yield increased by 790 kg/ha. Microbial inoculation restructured soil microbial community, influencing key taxa and interactions within the microbial network. A lignocellulose-degrading consortia consisting 37 genera was established, with a notable increase in the relative abundance of lignocellulose-degrading bacteria following inoculation. Among them, Pseudarthrobacter, with high lignin-degrading enzyme activity, emerged as a key genus after inoculation. Additionally, the abundance of lignin-degrading enzyme genes also increased significantly after inoculation. These findings offer new insights into how microbial inoculation accelerates the in situ decomposition of rice straw by reshaping the structure and function of lignocellulose-degrading consortia within the soil ecosystem.

Water retting process with hemp pre-treatment: effect on the enzymatic activities and microbial populations dynamic

Proper retting process of hemp stems, in which efficient separation of cellulose fiber from the rest of the stem is promoted by indigenous microorganisms able to degrade pectin, is essential for fiber production and quality. This research aimed to investigate the effect of a pre-treatment dew retting in field of hemp stalks on the pectinolytic enzymatic activity and microbiota dynamic during lab-scale water retting process. A strong increase in the pectinase activity as well as in the aerobic and anaerobic pectinolytic concentration was observed from 14 to 21 days, especially using hemp stalks that were not subjected to a pre-retting treatment on field (WRF0 0.690 ± 0.05 U/mL). Results revealed that the microbial diversity significantly varied over time during the water retting and the development of microbiota characterizing the water retting of hemp stalks of different biosystems used in this study was affected by pre-treatment conditions in the field and water retting process and by an interaction between the two methods. Although at the beginning of the experiment a high biodiversity was recorded in all biosystems, the water retting led to a selection of microbial populations in function of the time of pre-treatment in field, especially in bacterial populations. The use of hemp stems did not subject to a field pre-treatment seems to help the development of a homogeneous and specific pectinolytic microbiota with a higher enzymatic activity in respect to samples exposed to uncontrolled environmental conditions for 10, 20, or 30 days before the water retting process.Key points• Microbial diversity significantly varied over time during water retting.• Water retting microbiota was affected by dew pre-treatment in the field.• Retting of no pretreated hemp allows the development of specific microbiota with high enzymatic activity.

Be prepared: how does discontinuous hydration in Tabebuia heterophylla seeds induce stress tolerance in seedlings?

Discontinuous hydration and dehydration (HD) cycles refer to controlled imbibition followed by dehydration before seed germination. Here, we investigated whether the level of imbibition before HD cycles affects the physiology of Tabebuia heterophylla seeds and seedlings. Seeds were imbibed for 10 h (T1; phase I of imbibition) or 35 h (T2; phase II), dehydrated, and progressively rehydrated one to four times (HD cycles). Germination and biochemical parameters (membrane integrity; total soluble, reducing, and nonreducing (NRS) sugars; proteins, amino acids, proline, H2O2, catalase, ascorbate peroxidase, and glutathione reductase activity) were quantified at the last rehydration step of each cycle. Biometric and biochemical parameters (including pigments) were analysed in seedlings 60 days after germination. HD cycles at T1 led to reduced seed germination and greater plasma membrane damage, higher enzyme activity (catalase and glutathione reductase) and accumulation of NRS, total amino acids, and proline compared to the controls and T2 treatment. Cellular damage became more severe with more HD cycles. HD cycles at T2 synchronized germination regardless of the number of cycles and also had a priming effect. T2 seeds had less NRS, total amino acids, and proline content than T1. HD cycles at T1 produced seedlings with higher carotenoid and total chlorophyll content than controls and T2, while seedlings from HD cycles at T2 had higher amounts of osmoprotectants. HD cycles at T2 benefited seeds and seedlings more than at T1. This suggests that the physiological and biochemical effects of HD cycles in seeds modulate seedling plasticity, depending on water availability, potentially promoting increased tolerance to recurrent droughts that will be intensified with ongoing climate changes.

Genome-wide identification, molecular evolution, and functional characterization of fructokinase gene family in apple reveal its role in improving salinity tolerance

Fructokinase (FRK) is a regulator of fructose signaling in plants and gateway proteins that catalyze the initial step in fructose metabolism through phosphorylation. Our previous study demonstrated that MdFRK2 protein exhibit not only high affinity for fructose, but also high enzymatic activity due to sorbitol. However, genome-wide identification of the MdFRK gene family and their evolutionary dynamics in apple are yet to be reported. A systematic genome-wide analysis in this study identified a total of nine MdFRK gene members, which could phylogenetically be clustered into seven groups. Chromosomal location and synteny analysis of MdFRKs revealed that their expansion in the apple genome is primarily driven by tandem and segmental duplication events. Divergent expression patterns of MdFRKs were observed in four source-sink tissues and at five different apple fruit developmental stages, which suggested their potential crucial roles in the apple fruit development and sugar accumulation. Reverse transcription-quantitative PCR (RT-qPCR) identified candidate NaCl or drought stress responsive MdFRKs, and transgenic apple plants overexpressing MdFRK2 exhibited considerably enhanced salinity tolerance. Our results will be useful for understanding the functions of MdFRKs in the regulation of apple fruit development and salt stress response.

Rational Engineering of a β-Glucosidase (H0HC94) from Glycosyl Hydrolase Family I (GH1) to Improve Catalytic Performance on Cellobiose.

The conversion of lignocellulosic feedstocks by cellulases to glucose is a critical step in biofuel production. β-Glucosidases catalyze the final step in cellulose breakdown, producing glucose, and are often the rate-limiting step in biomass hydrolysis. The specific activity of most natural and engineered β-glucosidase is higher on the artificial substrate p-nitrophenyl β-d-glucopyranoside (pNPGlc) than on the natural substrate, cellobiose. We report an engineered β-glucosidase (Q319A H0HC94) with a 1.8-fold higher specific activity (366.3 ± 36 μmol/min/mg), a 1.5-fold increase in kcat (340.8 ± 27 s-1), and a 3-fold increase in catalytic efficiency (236.65 mM-1 s-1) over H0HC94 (WT) on cellobiose. Molecular dynamic simulations and protein structure network analysis indicate that the Q319A H0HC94 active site pocket is significantly remodeled compared to the WT, leading to changes in enzyme conformation, better accessibility of cellobiose inside the active site pocket, and higher enzymatic activity. This study shows the impact of rational engineering of a nonconserved residue to increase β-glucosidase substrate accessibility and catalytic efficiency by reducing crowding interaction between cellobiose and active site pocket residues near the gatekeeper region and increasing pocket volume and surface area. Thus, rational engineering of previously characterized enzymes could be an excellent strategy to improve cellulose hydrolysis.

Enhancing the inhibitory activities of polyphenols in passion fruit peel on α-Amylase and α-Glucosidase via β-Glucosidase-producing Lactobacillus fermentation

Passion fruit peel contained various phenolic compounds with α-glucosidase and α-amylase inhibitory activities in vitro. However, the bound state and glycosylated forms hindered their active expression. In this article, β-glucosidase-producing Lactobacilli were inoculated into fermented peels for targeted metabolism. The results showed that the release of bound polyphenols and the increase of free polyphenols were significantly correlated with β-glucosidase activity. The strain G1 with high enzyme activity degraded 48.20% of the bound polyphenols after 24 h. The ability of extracts to inhibit α-glucosidase and α-amylase activities was significantly enhanced and closely related to the content of free polyphenols (r = −0.96 and −0.88, P < 0.001). Computer-assisted screening identified 38 phenolic compounds as potential dual inhibitors of α-glucosidase and α-amylase. Among them, 22 α-glucosidase inhibitors and 24 α-amylase inhibitors were identified for the first time. Various inhibitors, including phenolic acids, flavones, and flavonols, increased in content after fermentation. The total content of 14 glycosylated polyphenols decreased to 28.38% at 48 h, which was opposite to the aglycone. Molecular docking indicated that glycosylated polyphenols bind to β-glucosidase through hydrogen bonds and van der Waals forces. Different glycosylations at the C-3 position of quercetin bound at same sites to β-glucosidase. This study contributes to the theory of enhancing the functional activity of natural products through targeted fermentation transformation.

Generation and characterization of two acid-resistant macrocin O-methyltransferase variants with a higher enzyme activity at 30 °C from Streptomyces fradiae

Tylosin is an important macrolide antibiotic produced by Streptomyces fradiae. In the biosynthesis of tylosin, macrocin O-methyltransferase TylF catalyzes the conversion of the side-product tylosin C (macrocin) to the primary component tylosin A (C/A conversion). This conversion is the rate-limiting step in the biosynthesis of tylosin, and affects the quality of the end product. To find a high activity and environment-adapted TylF enzyme, a TylF variant pool has been constructed via protein evolution approach in our previous study (Fan et al., 2023 [41]). In this study, the TylF variants with higher C/A conversion rates were expressed in E. coli and purified. The variants TylFY139F, TylFQ138H, F232Y and TylFT36S, V54A were shown to have a higher C/A conversion rate at 30 °C than that of TylF at 38 °C. Moreover, they had a greater acid resistance and showed more adaptable to the pH change during fermentation. Further protein structural and substrate-binding affinity analyses revealed that the T36S, V54A, Q138H, Y139F, and F232Y mutations enlarged the volume of the substrate-binding pocket, thereby increasing the affinity of enzyme variants for their substrates of SAM and macrocin, and decreasing the inhibition of SAH. Three of the TylF variants were overexpressed in the industrial tylosin-producing S. fradiae strain, and the recombinant strains showed the highest C/A conversion at 30 °C without heating up to 38 °C during the last 24 h of fermentation. This is of great energy-saving significance for tylosin industrial production.

Preparation of chiral polyether dendrimer and its non-bonding immobilized CALB for high enantioselective synergy resolution of amines

Monodisperse polyethylene glycol monomethyl ether and chiral glycidyl tosylate were utilized as substrates in the synthesis of the chiral monomer unit epoxypropyl polyethylene glycol monomethyl ether (MPEGn-EPO). The initiator propargyl-terminated polyethylene glycol alkyne-PEG3-OH was employed, while the phosphazene base P4-t-Bu facilitated the controlled ROP of MPEGn-EPO, resulting in clickable dendritic P(MPEGn-EPO) with terminal alkyne. The chiral four-arm dendritic polymer 4-Arm-PEG3-Mn was synthesized through a click reaction between 4-Arm-PEG3-N3 and P(MPEGn-EPO) using Cu(PPh3)3Br/CuBr as a catalyst. Candida antarctica lipase B (CALB) was chosen as a model enzyme for the preparation of a non-covalently immobilized biocatalyst (CALB-M) via a simple adsorption process with magnesium silicate and 4-Arm-PEG3-M4-20. The immobilized lipase CALB-M16 demonstrated high enzyme activity in the resolution of racemic amines, yielding chiral amines with enantioselectivity up to 99 % ee. Circular dichroism (CD) analysis indicated the formation of helical conformation in the dendritic polyethers 4-Arm-PEG3-M16-20 in the presence of magnesium silicate at 5 °C. The polyethylene glycol side groups of 4-Arm-PEG3-M16-20 are suggested to potentially form a crown ether-like structure with magnesium ions, which could enhance steric hindrance and induce a right-handed helix. Furthermore, the helical structure of this dendritic polyether is thought to work in synergy with CALB, improving the enantioselective resolution of amines.

Enhancing levan biosynthesis by destroying the strongly acidic environment caused by membrane-bound glucose dehydrogenase (mGDH) in Gluconobacter sp. MP2116

Levan produced by Gluconobacter spp. has great potential in biotechnological applications. However, Gluconobacter spp. can synthesize organic acids during fermentation, resulting in environmental acidification. Few studies have focused on the effects of environmental acidification on levan synthesis. This study revealed that the organic acids, mainly gluconic acid (GA) and 2-keto-gluconic acid (2KGA) secreted by Gluconobacter sp. MP2116 created a highly acidic environment (pH < 3) that inhibited levan biosynthesis. The levansucrase derived from strain MP2116 had high enzyme activity at pH 4.0 ∼ pH 6.5. When the ambient pH was less than 3, the enzyme activity decreased by 67 %. Knocking out the mgdh gene of membrane-bound glucose dehydrogenase (mGDH) in the GA and 2KGA synthesis pathway in strain MP2116 eliminated the inhibitory effect of high acid levels on levansucrase function. As a result, the levan yield increased from 7.4 g/l (wild-type) to 18.8 g/l (Δmgdh) during fermentation without pH control. This study provides a new strategy for improving levan production by preventing the inhibition of polysaccharide synthesis by environmental acidification.