Improving Lipid Production of Yarrowia lipolytica by the Aldehyde Dehydrogenase-Mediated Furfural Detoxification

Yarrowia lipolytica, the non-conventional yeast capable of high lipogenesis, is a microbial chassis for producing lipid-based biofuels and chemicals from renewable resources such as lignocellulosic biomass. However, the low tolerance of Y. lipolytica against furfural, a major inhibitory furan aldehyde derived from the pretreatment processes of lignocellulosic biomass, has restricted the efficient conversion of lignocellulosic hydrolysates. In this study, the furfural tolerance of Y. lipolytica has been improved by supporting its endogenous detoxification mechanism. Specifically, the endogenous genes encoding the aldehyde dehydrogenase family proteins were overexpressed in Y. lipolytica to support the conversion of furfural to furoic acid. Among them, YALI0E15400p (FALDH2) has shown the highest conversion rate of furfural to furoic acid and resulted in two-fold increased cell growth and lipid production in the presence of 0.4 g/L of furfural. To our knowledge, this is the first report to identify the native furfural detoxification mechanism and increase furfural resistance through rational engineering in Y. lipolytica. Overall, these results will improve the potential of Y. lipolytica to produce lipids and other value-added chemicals from a carbon-neutral feedstock of lignocellulosic biomass.     

Amine Transaminase Engineering for Spatially Bulky Substrate Acceptance

Amine transaminase (ATA) catalyzing stereoselective amination of prochiral ketones is an attractive alternative to transition metal catalysis. As wild‐type ATAs do not accept sterically hindered ketones, efforts to widen the substrate scope to more challenging targets are of general interest. We recently designed ATAs to accept aromatic and thus planar bulky amines, with a sequence‐based motif that supports the identification of novel enzymes. However, these variants were not active against 2,2‐dimethyl‐1‐phenyl‐propan‐1‐one, which carries a bulky tert‐butyl substituent adjacent to the carbonyl function. Here, we report a solution for this type of substrate. The evolved ATAs perform asymmetric synthesis of the respective R amine with high conversions by using either alanine or isopropylamine as amine donor.     

Production of furfural by acid hydrolysis of corncobs

Hydrolysis of corncobs for producing furfural was carried out in a pressurized batch reactor using superheated water and diluted sulphuric acid as catalyst. The range of experimental conditions was T=140–200°C and P=350–1550 kPa. Yields of furfural are reported as a function of reaction temperature, particle size, acid concentration and liquid/solid ratio. Attention has been focused on the solid residue remaining after the hydrolysis process, so that it can be further used as a humic fertilizer after oxiammoniation treatment.     

Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae

Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C₄–C₈) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94–96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source.     

A Scrutiny on the Reductive Amination of Carbonyl Compounds Catalyzed by Homogeneous Rh(I) Diphosphane Complexes

The reductive amination of a series of aldehydes with secondary amines and H₂ in the presence of a homogeneous Rh-diphosphane catalyst was studied in order to establish a general mechanism of this reaction and to identify conditions for the improvement of the amine/alcohol ratio in the product. Several possible intermediates as constituents of changing equilibria like half-aminals, N,O-acetals and aminals were observed in the reaction mixture by means of ¹H NMR spectroscopy. In individual trials, these compounds could be successfully hydrogenated under the conditions applied for reductive amination (50 bar H₂ pressure, MeOH). Some evidence is accumulated that half-aminals and N,O-acetals might be key intermediates of the reductive amination. Moreover, it was found that the formation of the undesired product alcohol is likely based on the reduction of the starting carbonyl compound. However, due to numerous equilibria consisting of several intermediates, general conclusions are hard to be drawn. Proof will be given that, in several cases, the efficiency of the reductive amination of aliphatic aldehydes can be significantly improved by prehydrogenation of the cationic [Rh(dppb)(COD)]⁺ complex.     

糠醛的水解制备和应用研究进展

糠醛是由可再生的生物质为原料转化得到的高价值化工产品，具有广阔的应用前景。本文综述了近年来生物质催化水解制备糠醛的研究进展，同时总结了糠醛衍生产品的制备和应用。在对半纤维素水解产糖反应和木糖脱水反应进行机理分析的基础上，从反应原料、溶剂体系、催化剂和分离方法等方面归纳总结了生物质催化水解制备糠醛的最新研究进展，并提出当前生物质制备糠醛方法中存在的问题和应对方案。在此基础上，分析了糠醛经氢化、胺化、氧化、缩醛化、聚合等反应获得高价值衍生产品的研究进展。提出要实现糠醛绿色高效的生产和应用，应着力设计低成本、低能耗、低污染且高效率的催化反应体系，同时推进重要糠醛衍生产品的综合高效利用。     

Reduction et amination electrochimiques du (Diethoxyphosphoryl)pyruvate d`ethyle

Study of the electrochemical reduction and amination mechanisms of ethylphosphonopyruvate allowed us to determine the best conditions for the preparations of the corresponding pinacol (EtOH, AcOH, AcOLi), alcohol (H₂O, pH = 4.5) and amine (H₂O, NH₃, NH₄Cl). The phosphonate group favours the solvation of the carbonyl and iminium functions. The electrochemical processes are controlled by the kinetics of the desolvation of the carbonyl group and the protonation of the enamine.     

Stereoselectivity of Four (R)‐Selective Transaminases for the Asymmetric Amination of Ketones

Four (R)‐ω‐transaminases originating from Hyphomonas neptunium (HN‐ωTA), Aspergillus terreus (AT‐ωTA) and Arthrobacter sp. (ArR‐ωTA), as well as an evolved transaminase (ArRmut11‐ωTA) were successfully employed for the amination of prochiral ketones leading to optically pure (R)‐amines. The first three transaminases displayed perfect stereoselectivity for the amination of all substrates tested (ee >99%). Furthermore, the transaminase AT‐ωTA led in most cases to better conversion than ArR‐ωTA and HN‐ωTA using D ‐alanine as amine donor. α‐Tetralone, which was the only substrate not accepted by HN‐ωTA, ArR‐ωTA, and AT‐ωTA, was successfully transformed with perfect enantioselectivity (ee >99%) into the corresponding optically pure amine employing the variant ArRmut11‐ωTA.     

The Evolution of an Amine Dehydrogenase Biocatalyst for the Asymmetric Production of Chiral Amines

The reductive amination of ketones to produce chiral amines is an important transformation in the production of pharmaceutical intermediates. Therefore, industrially applicable enzymatic methods that enable the selective synthesis of chiral amines could be very useful. Using a phenylalanine dehydrogenase scaffold devoid of amine dehydrogenase activity, a robust amine dehydrogenase has been evolved with a single two‐site library allowing for the direct production of (R)‐1‐(4‐fluorophenyl)‐propyl‐2‐amine from para‐fluorophenylacetone with a k_cat value of 6.85 s⁻¹ and a K_M value of 7.75 mM for the ketone substrate. This is the first example of a highly active amine dehydrogenase capable of accepting aliphatic and benzylic ketone substrates. The stereoselectivity of the evolved amine dehydrogenase was very high (>99.8% ee) showing that high selectivity of the wild‐type phenylalanine dehydrogenase was conserved in the evolution process. When paired with glucose/glucose dehydrogenase, NADH cofactor can be effficiently regenerated and the reaction driven to over 93% conversion. The broad specificity, high selectivity, and near complete conversion render this amine dehydrogenase an attractive target for further evolution toward pharmaceutical compounds and subsequent application.     

Asymmetric Bio‐amination of Ketones in Organic Solvents

ω‐Transaminases, employed as a lyophilised crude cell‐free extract, were successfully employed in organic solvent for the asymmetric amination of ketones without the need for immobilisation. Best activity was found for methyl tert‐butyl ether (MTBE) at a water activity of 0.6. The ω‐transaminases (9 different enzymes) accepted efficiently 2‐propylamine as amine donor when used in the solvent, which is not the case when they are used in aqueous solution. The bio‐amination in organic solvent showed several advantages such as higher reaction rates (up to 17‐fold), general acceptance of 2‐propylamine as amine donor, simple work‐up procedure (i.e., no basification and extraction required), easy recycling of the catalyst and lack of substrate inhibition. The biocatalysts maintained their excellent stereoselectivity in MTBE allowing the preparation of optically pure amines (ee >99%) with up to >99% conversion.     

磺基甜菜碱的合成研究进展

磺基甜菜碱具有性能温和、界面活性高、抗二价阳离子能力强等优点,被广泛应用于洗涤剂、化妆品、石油开采等领域。本文介绍了以胺为原料,经叔胺化-季铵化两步法合成磺基甜菜碱的合成工艺进展,重点阐述了制备叔胺中间体的4种方法,即卤化胺化法、醇一步催化胺化法、甲醛加氢法和羰基还原胺化法。从原料性能、收率、环保性、可操作性等方面进行了分析比较,讨论了各方法存在的问题及工业化的可行性。此外,对叔胺中间体的季铵化过程中所用试剂3-氯-2-羟基丙磺酸钠和磺酸内酯进行了比较,最后指出羰基还原胺化法是实现绿色工业化生产应该努力的方向,而3-氯-2-羟基丙磺酸钠是一种经济、绿色、环保的季铵化试剂。     

Computer Modeling Explains the Structural Reasons for the Difference in Reactivity of Amine Transaminases Regarding Prochiral Methylketones

Amine transaminases (ATAs) are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze the transfer of an amino group from an amino donor to an aldehyde and/or ketone. In the past decade, the enzymatic reductive amination of prochiral ketones catalyzed by ATAs has attracted the attention of researchers, and more traditional chemical routes were replaced by enzymatic ones in industrial manufacturing. In the present work, the influence of the presence of an α,β-unsaturated system in a methylketone model substrate was investigated, using a set of five wild-type ATAs, the (R)-selective from Aspergillus terreus (Atr-TA) and Mycobacterium vanbaalenii (Mva-TA), the (S)-selective from Chromobacterium violaceum (Cvi-TA), Ruegeria pomeroyi (Rpo-TA), V. fluvialis (Vfl-TA) and an engineered variant of V. fluvialis (ATA-256 from Codexis). The high conversion rate (80 to 99%) and optical purity (78 to 99% ee) of both (R)- and (S)-ATAs for the substrate 1-phenyl-3-butanone, using isopropylamine (IPA) as an amino donor, were observed. However, the double bond in the α,β-position of 4-phenylbut-3-en-2-one dramatically reduced wild-type ATA reactivity, leading to conversions of <10% (without affecting the enantioselectivity). In contrast, the commercially engineered V. fluvialis variant, ATA-256, still enabled an 87% conversion, yielding a corresponding amine with >99% ee. Computational docking simulations showed the differences in orientation and intermolecular interactions in the active sites, providing insights to rationalize the observed experimental results.     

Lewis Acid–Lewis Base‐Catalysed Enantioselective Addition of α‐Ketonitriles to Aldehydes

Additions of structurally diverse α‐ketonitriles to aromatic and aliphatic prochiral aldehydes yielding highly enantioenriched acylated cyanohydrins were achieved using a combination of a titanium salen dimer and an achiral or chiral Lewis base. In most cases high yields and high enantioselectivities were observed. The ee was moderate in the initial part of the reaction but increased over time. This could be avoided, and higher ees obtained, by keeping the titanium complex, in the presence or absence of aldehyde and ketonitrile, at −40 °C prior to the addition of the Lewis base. A mechanism initiated by nucleophilic attack of the tertiary amine at the carbonyl carbon atom of the ketonitile is supported by ¹³C labelling experiments.     

Preparation of highly phenol substituted bio-oil–phenol–formaldehyde adhesives with enhanced bonding performance using furfural as crosslinking agent

Highly phenol substituted bio-oil–phenol–formaldehyde (BPF) adhesives were prepared via the phenolization-copolymerization method, in which furfural was used as a novel crosslinking agent to improve the bonding performance. The effects of bio-oil percentage, furfural content, curing temperature, and pressure on the performance of BPF adhesives have been studied in detail. A BPF adhesive with 75% bio-oil percentage and 15% furfural loading (named 75BPF_15) was synthesized, which was cured at 180 °C and 4 MPa. Compared to the conventional phenol–formaldehyde adhesive, the wet tensile strength of 75BPF_15 adhesive reached 2.84 MPa, which was significantly higher than the 1.54 MPa of PF. A possible mechanism of BPF adhesives crosslinked with furfural is proposed. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 46995.     

Selective Formation of Formamidines or 7‐Aminomethylbenzoxazoles from Unprecedented Couplings between Benzoxazoles and Amines

A method for the selective synthesis of amidines by ring‐opening amination or of 7‐aminomethylbenzoxazoles via a novel three‐component‐coupling reaction (arene, dichloromethane and amine) from benzoxazoles and amines was developed. Amidines could be further converted into 2‐aminobenzoxazoles in high yield.     

Production of xylose,furfural, fermentable sugars and ethanol from agricultural residues

With the developing shortage of petroleum, reliance on biomass as a source of chemicals and fuels will increase. In the present work, bagasse and rice husk were subjected to dilute acid (H₂SO₄) hydrolysis using pressurised water to obtain furfural and fermentable sugars. Various process conditions such as particle size, solid-liquid ratio, acid concentration, reaction time and temperature have been studied to optimise yields of furfural, xylose and other fermentable sugars. The use of particle sizes smaller than 495 μm did not further increase the yield of reducing sugars. A solid-liquid ratio of 1:15 was found to be the most suitable for production of reducing sugars. Hydrolysis using 0.4% H₂SO₄ at 453 K resulted in selective yields (g per 100 g of dried agricultural residues) of xylose from bagasse (22.5%) and rice husk (21.5%). A maximum yield of furfural was obtained using 0.4% H₂SO₄ at 473 K from bagasse (11.5%) and rice husk (10.9%). It was also found that hydrolysis using 1% H₂SO₄ at 493 K resulted in maximum yields of total reducing sugar from bagasse (53.5%) and rice husk (50%). The reducing sugars obtained were fermented to ethanol after removal of furfural. The effect of furfural on the fermentation of sugars to ethanol was also studied. Based on these studies, an integrated two-step process for the production of furfural and fermentable sugars could be envisaged. In the first step, using 0.4% H₂SO₄ at 473 K, furfural could be obtained, while in the second step, the use of 1% H₂SO₄ at 493 K should result in the production of fermentable sugars.     

Determination of Furfural and Hydroxymethylfurfural Formed From Biomass Under Acidic Conditions

Abstract

A rapid method for simultaneous determination of furfural and hydroxymethylfurfural (HMF) in the filtrate of an acidic treatment of biomass was developed based on UV spectrophotometer. Interference from acid soluble lignin is alleviated by the use of absorbance difference spectrum before and after reduction with sodium borohydride (NaBH₄). The concentrations of furfural and HMF in the filtrate are determined by measuring the absorbance of the difference spectrum at 277 nm and 285 nm, the characteristic absorption maxima for furfural and HMF, respectively. Two simultaneous equations are solved to obtain the concentrations of furfural and HMF in the filtrate. Acid soluble lignin in the solution is determined to cause negligible interference on the analysis.     

Preparation of tannin–formaldehyde–furfural resin with pretreatment of depolymerization of condensed tannin and ring opening of furfural

As renewable and economical bio-based resources, tannin can polymerize with aldehyde to form resin, furfural can partially substitute formaldehyde for the preparation of resin with phenols. Aiming to enhance the reaction efficiency between tannin and furfural by weakening the steric hindrance, a pretreatment was applied to depolymerize the macromolecule of tannin into small one and open the furan ring structure of furfural before the preparation of tannin–formaldehyde–furfural (TFFu) resin. Modified furfural was used to partially substitute formaldehyde by the ratios of 10, 20, and 30%. Chemical structure characteristics, thermal behaviors, dynamic viscosity, and fracture surface micrograph of the TF and TFFu resins were analyzed by Fourier transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analysis, rheological measurement, and scanning electron microscopy, respectively. The result indicated that co-polycondensation with modified furfural decreased the free formaldehyde content of TFFu resin, and increased the thermal stability. Moreover, when the formaldehyde was substituted by modified furfural in 10%, the TFFu resin had the most smooth and compact fracture surface.     

A novel injectable and in situ crosslinked hydrogel based on hyaluronic acid and α,β-polyaspartylhydrazide

A new injectable and in situ crosslinked hydrogel, based on a hyaluronic acid (HA) derivative and α,β-polyaspartylhydrazide (PAHy), was produced during the research. This biodegradable and high molecular weight hydrogel can be in situ crosslinked in 15 s by the condensation reaction between aldehyde and amine groups. The HA derivative carrying aldehyde (HAALD) was oxidized from HA by sodium periodate, while the synthesis of hydrogel was performed in a phosphate-buffered saline solution (PBS) using HAALD and PAHy without addition of crosslinker or catalyst. The pH and concentration of the reaction solution, considered as the important factors of the amine-aldehyde reaction, were changed to reveal the crosslinking rule. Thereafter, crosslinked hydrogels were characterized by gelation time, gel content, swelling ratio, and in vitro degradation. Furthermore, the modified polymers were characterized by Fourier transformed infrared (FTIR) spectroscopy to examine their structures. Results from scanning electron microscope (SEM) observations confirmed that a freeze-dried sample revealed a porous hydrogel structure with interconnected pores. The measurement of the cell adhesion confirmed the application potential of HAALD-PAHy hydrogels. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Reductive Amination Without the Aldehyde: Use of a Ketolactol as an Aldehyde Surrogate

To overcome stability issues associated with the use of an aldehyde in a catalytic reductive amination reaction, a cyclic ketolactol (ω-hydroxylactone) was employed as an aldehyde surrogate to form a γ-aminoacid. The reaction proceeded most favorably over a Pt/C catalyst. The thermodynamics of each step were evaluated using density functional theory calculations, which correctly predicted the dominance of the ring-closed lactol reactant, yet suggested a preference for a ring-opened iminium intermediate upon the initial, slightly endoergic addition of amine substrate. Exoergic hydrogenation of this intermediate provided the thermodynamic driving force for the overall transformation. During development, the reaction was observed to depend significantly on the volumetric gas to liquid mass transfer coefficient (k_La) and this parameter was optimized to ensure successful scale up in a 400 L stirred tank reactor.