Preface for special issue on chemical bioproduction / 生物工程学报

Engineering efficient enzymes or microbial cell factories should help to establish green bio-manufacturing process for chemical overproduction. The rapid advances and development in synthetic biology, systems biology and enzymatic engineering accerleate the establishing feasbile bioprocess for chemical biosynthesis, including expanding the chemical kingdom and improving the productivity. To consolidate the latest advances in chemical biosynthesis and promote green bio-manufaturing, we organized a special issue on chemical bioproduction that including review or original research papers about enzymatic biosynthesis, cell factory, one-carbon based biorefinery and feasible strategies. These papers comprehensively discussed the latest advaces, the challenges as well as the possible solutions in chemical biomanufacturing.

Research progress on application of multi-enzyme-catalyzed cascade reactions in enzymatic synthesis of natural products / 中国中药杂志

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.

Protopanaxadiol-type ginsenoside hydrolases and their application in the preparation of ginsenoside Compound K: a review / 生物工程学报

Ginsenoside Compound K (CK) has anti-cancer and anti-inflammatory pharmacological activities. It has not been isolated from natural ginseng and is mainly prepared by deglycosylation of protopanaxadiol. Compared with the traditional physicochemical preparation methods, the preparation of CK by hydrolysis with protopanaxadiol-type (PPD-type) ginsenoside hydrolases has the advantages of high specificity, environmental-friendliness, high efficiency and high stability. In this review, the PPD-type ginsenoside hydrolases were classified into three categories based on the differences in the glycosyl-linked carbon atoms of the hydrolase action. It was found that most of the hydrolases that could prepare CK were PPD-type ginsenoside hydrolase type Ⅲ. In addition, the applications of hydrolases in the preparation of CK were summarized and evaluated to facilitate large-scale preparation of CK and its development in the food and pharmaceutical industries.

Role of nanomaterials in catalytic reduction of organic pollutants

Developing innovative technologies for the effective treatment of wastewater containing organic pollutants is of extreme importance across the globe. The organic pollutants such as dyes and nitrophenols are the common hazardous pollutants known for their adverse effects on humans and aquatic organisms. Various methods have been used for the removal of organic pollutants from wastewater but they suffer limitations such as high cost, time consuming removal process and production of sludge or toxic by-products. In recent years, chemical reduction method is becoming popular for removal of organic pollutants using various nanomaterials as catalysts. Nanomaterials show great potential for removal of organic pollutants due to large surface area which provides high catalytic activity. In the present review, current studies on catalytic reduction of organic pollutants (dyes and nitrophenols) using four different types of nanomaterials specifically carbon nanotubes, silica, metal oxide and chitosan polymer based have been explored. The factors affecting the catalytic process and mechanism of catalysis is explained in detail.In addition, a critical discussion about the pros and cons of each nano-catalyst have also been included for developing better understanding of the choice of catalyst.

Characterization of highly active tyrosine ammonia lyase and its application in biosynthesis of p-coumaric acid / 生物工程学报

p-coumaric acid is one of the aromatic compounds that are widely used in food, cosmetics and medicine due to its properties of antibacterium, antioxidation and cardiovascular disease prevention. Tyrosine ammonia-lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid. However, the lack of highly active and specific tyrosine ammonia lyase limits cost-effective microbial production of p-coumaric acid. In order to improve biosynthesis efficiency of p-coumaric acid, two tyrosine ammonia-lyases, namely Fc-TAL2 derived from Flavobacterium columnare and Fs-TAL derived from Flavobacterium suncheonense, were selected and characterized. The optimum temperature (55 ℃) and pH (9.5) for Fs-TAL and Fc-TAL2 are the same. Under optimal conditions, the specific enzyme activity of Fs-TAL and Fc-TAL2 were 82.47 U/mg and 13.27 U/mg, respectively. Structural simulation and alignment analysis showed that the orientation of the phenolic hydroxyl group of the conserved Y50 residue on the inner lid loop and its distance to the substrate were the main reasons accounting for the higher activity of Fs-TAL than that of Fc-TAL2. The higher activity and specificity of Fs-TAL were further confirmed via whole-cell catalysis using recombinant Escherichia coli, which could convert 10 g/L tyrosine into 6.2 g/L p-coumaric acid with a yield of 67.9%. This study provides alternative tyrosine ammonia-lyases and may facilitate the microbial production of p-coumaric acid and its derivatives.

Enzyme-instructed hybrid nanogel/nanofiber oligopeptide hydrogel for localized protein delivery

Enzyme-catalysis self-assembled oligopeptide hydrogel holds great interest in drug delivery, which has merits of biocompatibility, biodegradability and mild gelation conditions. However, its application for protein delivery is greatly limited by inevitable degradation of enzyme on the encapsulated proteins leading to loss of protein activity. Moreover, for the intracellularly acted proteins, cell membrane as a primary barrier hinders the transmembrane delivery of proteins. The internalized proteins also suffer from acidic and enzymatic degradation in endosomes and lysosomes. We herein develop a protease-manipulated hybrid nanogel/nanofiber hydrogel for localized delivery of intracellularly acted proteins. The embedded polymeric nanogels (CytoC/aNGs) preserve activity of cytochrome

Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid / 生物工程学报

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.

Application of immobilized glycosidase in the synthesis of glycoside compounds / 生物工程学报

Glycoside compounds are widely used in medicine, food, surfactant, and cosmetics. The glycosidase-catalyzed synthesis of glycoside can be operated at mild reaction conditions with low material cost. The glycosidase-catalyzed processes include reverse hydrolysis and transglycosylation, appropriately reducing the water activity in both processes may effectively improve the catalytic efficiency of glucosidase. However, glucosidase is prone to be deactivated at low water activity. Thus, glucosidase was immobilized to maintain its activity in the low water activity environment, and even in neat organic solvent system. This article summarizes the advances in glycosidase immobilization in the past 30 years, including single or comprehensive immobilization techniques, and immobilization techniques combined with genetic engineering, with the aim to provide a reference for the synthesis of glycosides using immobilized glycosidases.

Bio-based molecules for biosynthesis of nano-metallic materials / 生物工程学报

Nano-metallic materials are playing an important role in the application of medicine, catalysis, antibacterial and anti-toxin due to their obvious advantages, including nanocrystalline strengthening effect, high photo-absorptivity, high surface energy and single magnetic region performance. In recent years, with the increasing consumption of global petrochemical resources and the aggravation of environmental pollution, nanomaterials based on bio-based molecules have aroused great concern. Bio-based molecules refer to small molecules and macromolecules directly or indirectly derived from biomass. They usually have good biocompatibility, low toxicity, degradability, wide source and low price. Besides, most bio-based molecules have unique physical, chemical properties and physiological activity, such as optical activity, acid/alkali amphoteric property, hydrophilic property and easy coordination with metal ions. Thus, the corresponding nano-materials based on bio-based molecules also have unique functions, such as anti-inflammatory, anti-cancer, anti-oxidation, antiviral fall blood sugar and blood fat etc. In this paper, we give a comprehensive overview of the preparation and application of nano-metallic materials based on bio-based molecules in recent years.

Photo Catalytic Silver Modified Orthodontic Brackets - An Innovative Method for Prevention of White Spot Lesions

Association between fixed orthodontic therapy and enamel decalcification causing periodontal disease and enamel decalcification is a known problem of orthodontic treatment. The root cause of this is the fact that brackets provide an ideal environment for bacteria to accumulate and multiply. Hence modifying the surface of brackets with some photocatalytic antibacterial substance could help in prevention of this side-effect. The current study was thus planned to evaluate the anti-adherence of bacteria to photocatalytic silver coated brackets for the prevention of white spot lesions. METHODSAfter obtaining ethical clearance from the institutional ethics committee, 40 metal brackets of upper central incisor were taken. These brackets were divided into group 1 & group 2 each containing twenty brackets. Group 1 (control group) consisted of plain metal brackets while group 2 (experimental group) consisted of silver coated brackets. Both the groups were subjected to laboratory bacterial tests to assess the bacterial adhesion to brackets and then statistical analysis was done to obtain results. RESULTSSilver modified brackets showed around 25 % less adhesion of bacteria as compared with plain brackets. CONCLUSIONSModifying orthodontic brackets by coating them with photocatalytic silver could prove to be an innovative and effective method in prevention of white spot lesions after fixed orthodontic therapy.

Nanoestructuras de sílice, con diámetro y distribución de mesoporos variable, modificadas con ácido tungstofosfórico como catalizadores en la síntesis de quinoxalinas / Silica nanostructures, with variable diameter and mesopore size distribution, modified with tungstophosphoric acid as catalyst in the synthesis of quinoxalines / Nanoestruturas de sílica, com distribuição variável de diâmetro e tamanho de mesoporos, modificadas com ácido tungstofosfórico como catalisador na síntese de a síntese da quinoxalina

Resumen Se sintetizaron catalizadores (SNX#WPA) basados en ácido tungstofosfórico, en soportes de nanoestructuras de sílice (SNX), con distribución de diámetros y tamaños de mesoporos variables. Las SNX se prepararon en medio de octano/agua, usando poliestireno y bromuro de cetiltrimetilamonio como plantillas. Los materiales se caracterizaron por DRX, TEM y adsorción/desorción de nitrógeno. La relación octano/agua influyó tanto en la morfología y el tamaño de las SNX como en la distribución del tamaño de poro. Las SNX obtenidas utilizando relaciones OCT/H2O en el rango de 0,07-0,35, presentan mesoporos pequeños (5-6 nm) y grandes (28-34 nm), generados principalmente por poliestireno. Los mesoporos grandes y su contribución de volumen fueron claramente más altos que en las muestras SN1, SN2 y SN3. La estructura y la morfología de SNX#WPA fueron similares a las de las SNX usadas como soporte. Además, la caracterización de todos los materiales SNX#WPA por FT-IR y 31P NMR indicó la presencia de especies [PW12O40]3- y [H3-XPW12O40](3-X)- sin degradar. Según los resultados de la valoración potenciométrica, los sólidos presentaron sitios ácidos muy fuertes. Se evaluó la actividad de SNX#WPA como catalizadores en la síntesis de quinoxalinas, a partir de lo cual se obtuvieron altos rendimientos, sin formación de subproductos. De ello resultó que los materiales preparados son catalizadores altamente selectivos y reutilizables.

Abstract Tungstophosphoric acid supported on silica nanostructures (SNX#WPA) with variable diameter and mesopore size distribution were synthetized. Silica nanostructures (SNX) were prepared in octane/aqueous media using polystyrene and CTAB as organic templates. The materials were characterized by XRD, SEM, TEM and dinitrogen adsorption/ desorption isotherm analysis. The octane/ water ratio influenced the morphology and size of SNX prepared, as well as its pore size distribution. The SNX samples obtained using OCT/H2O ratios in the range 0.07-0.35 (SN4, SN5, and SN6 samples). present small (5-6 nm) and large (28-34 nm) mesopores (mainly generated by polystyrene). Large mesopores and their volume contribution were clearly higher than in the SN1, SN2, and SN3 samples. The structure and morphology of SNX#WPA samples were similar to those of the SNX. Furthermore, the characterization of all the SNX#WPA materials by FT-IR and 31P NMR indicated the presence of undegraded [PW12O40]3- and [H3-XPW12O40](3-X)-species. According to the potentiometric titration results, the solids presented very strong acid sites. The performance of SNX#WPA materials as catalysts in the synthesis of quinoxalines was evaluated. The yields achieved were high, without formation of by-products resulting from competitive reactions or decomposition products, so the prepared materials are highly selective and reusable catalysts.

Resumo Catalisadores (SNX#WPA) baseados em ácido tungsofosfórico suportado em nanoestruturas de sílica (SNX) foram sintetizados com distribuição de tamanhos e diâmetros variados de mesoporos. Os SNX foram preparados em meio octano/aquoso usando poliestireno e brometo de cetiltrimetilamônio como modelos orgânicos. Os materiais foram caracterizados por DRX, TEM e adsorção/ dessorção de nitrogénio. A razão octano/ agua influenciou a morfologia e o tamanho do SNX, bem como a distribuição do tamanho dos poros. O SNX obtido usando razões OCT/ H2O na faixa de 0,07-0,35, possui mesoporos pequenos (5-6 nm) e grandes (28-34 nm) (gerados principalmente por poliestireno). Mesoporos grandes e sua contribuição em volume foram claramente maiores do que nas amostras SN1, SN2 e SN3. A estrutura e a morfologia do SNX#WPA foram semelhantes às do SNX usado como suporte. Além disso, a caracterização de todos os materiais SNX#WPA por FT-IR e 31P NMR indicou a presença das espécies [PW12O40]3- e [H3-XPW12O40](3-X)- sem degradar. De acordo com os resultados da titulação potenciométrica, os sólidos apresentaram locais ácidos muito fortes. A atividade do SNX#WPA como catalisadores na síntese de quinoxalinas foi avaliada, produzindo altos rendimentos, sem formação de subprodutos, resultando em materiais catalisadores altamente seletivos e reutilizáveis.

Effect of ions and inhibitors on the catalytic activity and structural stability of S. aureus enolase

Constantly rising energy demands, finite fossil fuel reserves and deteriorating environmental conditions have invokedworldwide interest to explore the sustainable sources of renewable biofuels. Locally adapted photosynthetic oleaginousmicroalgae with rapid growth on variable temperatures could be an ideal way for bioremediating the wastewater (WW) whileproducing the feedstock for biodiesel. To test this notion, an unknown strain was isolated from a sewage fed lake (Neela-Hauz).It was discerned as Chlorella sorokiniana-I using the 16S rDNA and 18S rDNA barcodes. The culture conditions such as pH,illumination, different temperature ranges and growth medium were cohesively optimized prior to the assessment of C.sorokiniana-I’s efficacy to remediate the WW and biodiesel production. The strain has thrived well up to 40C when continuously grown for 15 days. The highest lipid accumulation and biomass productivity were recorded in 100% WW. Fatty acidmethyl ester (FAME) content was observed to be more than twice in WW (47%), compared to control synthetic media, TAP(20%) and BG11 (10%), which indicate the importance of this new isolate for producing economically viable biodiesel.Moreover, it is highly efficient in removing the total nitrogen (77%), total phosphorous (81%), iron (67%) and calcium (42%)from the WW. The quality of WW was considerably improved by reducing the overall chemical oxygen demand (48%),biological oxygen demand (47%) and alkalinity (15%). Thus, C. sorokiniana-I could be an ideal alga for the tropical countriesin the remediation of WW while producing feedstock for biodiesel in a cost-effective manner.

Enhancing sensitivity of phenol-sulfuric acid assay using orthophosphoric acid as modifier

Background:Phenol-sulfuric acid assay has been widely used for quantification of sugars in biological fluid and industry. The conventional method originally proposed by Dubois et al, was modified several times for enhancing sensitivity of assay such as substituting phenol with other chromogens, by optimizing assay conditions and by adding phenol after dehydration reaction. Both conventional and modified assays have utilized acid catalysis property of sulphuric acid but effect of adding phosphoric acid has not been studied before. Methods:The present study was conducted in Department of Biochemistry, ANIIMS, Port Blair and IGIMS, Patna. The method being developed in our study consisted of adding orthophosphoricacid to the reaction mixture before addition of sulphuric acid. The method was optimized with different amounts of orthophosphoric acid. Statistical analysis was done using SPSS and Microsoft Excel software.Results:The current study found an enhancingeffect (on sensitivity) of orthophosphoric acid in optimal concentration of 5.16 mmoles. Comparison among standard curves of methods that were compared showed that the curve was steepest for current study and average absorbance was 0.199±0.017 for conventional, 0.253±0.011 for method by Rasouli et al,and 0.290±0.013 for current study. Pooled serum analysis exhibited absorbance of 0.157±0.015 in conventional method while in modified conventional method absorbance was 0.234±0.010 and highest absorbance was observed in current study at 0.281±0.012.Conclusions:Our results suggest that orthophosphoric acid exerts a positively modifying effect on phenol sulphuric acid assay.

Catalysis of enzymes under industrial environment and their adaptive modifications: a review / 生物工程学报

Enzymes have a wide range of applications and great industrial potential. However, large-scale applications of enzymes are restricted by the harsh industrial environment, such as high temperature, strong acid/alkali, high salt, organic solvents, and high substrate concentration. Adaptive modification (such as rational or semi-rational design, directed evolution and immobilization) is the most common strategy to improve the catalysis of enzymes under industrial conditions. Here, we review the catalysis of enzymes in the industrial environment and various methods adopted for the adaptive modifications in recent years, to provide reference for the adaptive modifications of enzymes.

A novel chlorpyrifos hydrolase CPD from Paracoccus sp. TRP: molecular cloning, characterization and catalytic mechanism

Background: Biodegradation is a reliable approach for efficiently eliminating persistent pollutants such as chlorpyrifos. Despite many bacteria or fungi isolated from contaminated environment and capable of degrading chlorpyrifos, limited enzymes responsible for its degradation have been identified, let alone the catalytic mechanism of the enzymes. Results: In present study, the gene cpd encoding a chlorpyrifos hydrolase was cloned by analysis of genomic sequence of Paracoccus sp. TRP. Phylogenetic analysis and BLAST indicated that CPD was a novel member of organophosphate hydrolases. The purified CPD enzyme, with conserved catalytic triad (Ser155-Asp251-His281) and motif Gly-Asp-Ser-Ala-Gly, was significantly inhibited by PMSF, a serine modifier. Molecular docking between CPD and chlorpyrifos showed that Ser155 was adjacent to chlorpyrifos, which indicated that Ser155 may be the active amino acid involved in chlorpyrifos degradation. This speculation was confirmed by site-directed mutagenesis of Ser155Ala accounting for the decreased activity of CPD towards chlorpyrifos. According to the key role of Ser155 in chlorpyrifos degradation and molecular docking conformation, the nucleophilic catalytic mechanism for chlorpyrifos degradation by CPD was proposed. Conclusion: The novel enzyme CPD was capable of hydrolyze chlorpyrifos and Ser155 played key role during degradation of chlorpyrifos.

Carbon dioxide/methanol conversion cycle based on cascade enzymatic reactions supported on superparamagnetic nanoparticles

ABSTRACT The conversion of carbon dioxide into important industrial feedstock is a subject of growing interest in modern society. A possible way to achieve this goal is by carrying out the CO2/methanol cascade reaction, allowing the recycle of CO2 using either chemical catalysts or enzymes. Efficient and selective reactions can be performed by enzymes; however, due to their low stability, immobilization protocols are required to improve their performance. The cascade reaction to reduce carbon dioxide into methanol has been explored by the authors, using, sequentially, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FalDH), and formate dehydrogenase (FDH), powered by NAD+/NADH and glutamate dehydrogenase (GDH) as the co-enzyme regenerating system. All the enzymes have been immobilized on functionalized magnetite nanoparticles, and their reactions investigated separately in order to establish the best performance conditions. Although the stepwise scheme led to only 2.3% yield of methanol per NADH; in a batch system under CO2 pressure, the combination of the four immobilized enzymes increased the methanol yield by 64 fold. The studies indicated a successful regeneration of NADH in situ, envisaging a real possibility of using immobilized enzymes to perform the cascade CO2-methanol reaction.

Controlled synthesis of noble metal nanomaterials: motivation, principles, and opportunities in nanocatalysis

ABSTRACT This review describes some principles of the controlled synthesis of metal nanoparticles, focusing on how the fundamental understanding of their synthesis in the solution-phase can be put to tailor size, shape, composition, and architecture. The maneuvering over these parameters not only enable the tuning of properties, but also the maximization and optimization of performances for various applications. Herein, we start with a brief description of metallic nanoparticles, highlighting the motivation for achieving physicochemical control in their synthesis. After that, we turn our attention to some important definitions and classifications as well as their unique properties such as surface and quantum effects. Moreover, we discuss the strategies for the controlled synthesis of metal nanomaterials based on the top-down and bottom-up approaches, focusing our discussion on their formation mechanisms in liquid-phase in terms of both thermodynamic and kinetic control. Finally, we point out the promising applications of controlled nanomaterials in the field of nanocatalysis and plasmon-enhanced catalysis, describing some of the current challenges in these fields.

Enzymatic synthesis of three kinds of galactose-cholesterol ligands and their structure-activity relationship with liver targeting / 中草药

Objective To construct three kinds of doxorubicin liposomes modified with cholesterol-galactose ligand by lipase-catalyzed method and compare their characteristic of pharmacokinetics and tissue distribution in vivo. Methods Three types of cholesterol-galactose ligands, CHS-C8-GalNAc, CHS-C8-GAL, and CHS-C8-LA were synthetized by lipase-catalyzed method in nonaqueous phase. The structure characterizations of products were obtained by MS and NMR. Conventional liposomes (CL DOX) and ligand-coupled liposomes (NGal-LP DOX, Gal-LP DOX, and LA-LP DOX) were prepared by thin film dispersion-ammonium sulphate gradient method. Structure-activity relationship between asialoglycoprotein receptor (ASGPr) and the chemical structure of the glycolipids was explored through the pharmacokinetics and tissue distribution parameters of ligand-coupled liposomes in vivo. Results The desired compounds with a high yield of above 90% were confirmed by MS and NMR. The liposomes average size was lower than 90 nm, polymer dispersity index was lower than 0.1, encapsulation efficiency was greater than 99%, leakage rat was lower than 5% with 24 h, and zeta potential closed to zero. The affinity of the three ligand molecules to liver was the following order: CHS-C8-GalNAc > CHS-C8-LA > CHS-C8-Gal. However, only the liposomes modified with CHS-C8-GalNAc could significantly be inhibited by the preinjection of asialofetuin for hepatic uptake rate (P 0.05). Conclusion The ligand with N-acetylgalactosamine residue showed high targeting efficiency for hepatocytes, while the ligand with D-galactose (Gal) or lactitol residue could competitive bind with Gal particle receptor on kupffer cells.

Enzymatic synthesis of xylulose from formaldehyde / 生物工程学报

Xylulose as a metabolic intermediate is the precursor of rare sugars, and its unique pattern of biological activity plays an important role in the fields of food, health, medicine and so on. The aim of this study was to design a new pathway for xylulose synthesis from formaldehyde, which is one of the most simple and basic organic substrate. The pathway was comprised of 3 steps: (1) formaldehyde was converted to glycolaldehyde by benzoylformate decarboxylase mutant BFD-M3 (from Pseudomonas putida); (2) formaldehyde and glycolaldehyde were converted to dihydroxyacetone by BFD-M3 as well; (3) glycolaldehyde and dihydroxyacetone were converted to xylulose by transaldolase mutant TalB-F178Y (from Escherichia coli). By adding formaldehyde (5 g/L), BFD-M3 and TalB-F178Y in one pot, xylulose was produced at a conversion rate of 0.4%. Through optimizing the concentration of formaldehyde, the conversion rate of xylulose was increased to 4.6% (20 g/L formaldehyde), which is 11.5 folds higher than the initial value. In order to further improve the xylulose conversion rate, we employed Scaffold Self-Assembly technique to co-immobilize BFD-M3 and TalB-F178Y. Finally, the xylulose conversion rate reached 14.02%. This study provides a new scheme for the biosynthesis of rare sugars.

Expression, purification, characterization and application of α-amino acid ester acyltransferase from recombinant Escherichia coli / 生物工程学报

α-Amino acid ester acyltransferase (Aet) catalyzes the L-alanyl-L-glutamine forming reaction from L-alaine methylester hydrochloride and L-glutamine. In this study, the recombinant Escherichia coli saet-QC01 was used to express the α-amino acid acyltransferase, and its expression conditions were optimized. The recombinant protein was separated and purified by Ni-NTA affinity chromatography, and its enzymatic properties and catalytic applications were studied. The induction conditions suitable for enzyme production optimized were as follows: The temperature was 20 ℃, the induction stage (OD₆₀₀=2.0-2.5), IPTG concentration was 0.6 mmol/L, induction time was 12 h. The optimal reaction conditions of α-amino acid acyltransferase were 27 ℃, pH 8.5, it was most stable between pH 7.0 and 8.0 and relatively stable in an acidic environment, and low concentration of Co²⁺ or EDTA could promote the enzyme activity. Under optimal reaction conditions, 600 mmol/L of L-alaine methylester hydrochloride and 480 mmol/L of L-glutamine, the yield of L-alanyl-L-glutamine reached 78.2 g/L and productivity of 1.955 g/L/min, the conversion rate reached 75.0%. α-Amino acid ester acyltransferase has excellent acid-basei resistance, high catalytic efficiency. These characteristics suggest its application prospects in the industrial production.