论文总字数：42551字

2020 届毕业设计（论文）

题 目： Biotransformation from HMF to FDCA

专 业： Pharmaceutical Science

班 级：1601

姓 名： IDRISSOV TIMUR

指导老师：张志刚

起讫日期：2020年4月-2020年6月

2020年05月

Title: Biotransformation from HMF to FDCA

Prepared by: Idrissov Timur
Class: Pharmacy 1601
Student ID: L1001160628

Introduction

Renewable raw energy sources in prospective industries have to be established with the depletion of fossil fuels. Biomass is an abundant and cheap resource, and can be a potential candidate for various chemicals to be made. The 5- Hydroxymethylfurfural (HMF), obtainable from the dehydration of carbohydrates derived from lignocellulose, is a promising chemical platform for the development of bio-based chemicals. HMF, for example, can be oxidised to 2,5-furandicarboxylic acid (FDCA). FDCA is an important chemical platform and is one of the top 12 value-added chemicals from biomass according to the US study. FDCA is used in many fields, and replacing oil-derived terephthalate with poly(ethylene terephthalate) (PET) synthesis is the most promising application; this method represents a new way of producing biomass-derived polyester to replace the oil-based polyester in whole or in part. Poly(ethylene 2,5-furandicarboxylate) (PEF), an ethylene glycol and FDCA esterification drug, is comparable to poly(ethylene terephthalate) (PET) in beer, soft drink, and water packaging materials.   Chemical reactions to oxidation are usually performed at high temperature and high pressure. Biocatalytic processing is normally done under relatively mild conditions as opposed to chemical processes, which generally requires fewer and less toxic chemicals. In contrast, biocatalytic reactions have several advantages , such as high precision, low toxicity and a more environmentally desirable footprint. Numerous enzymatic reactions to synthesize FDCA from HMF have been documented using various oxidases such as HMF oxidase, fungal aryl-alcohol oxidase, non-specific peroxygenase, chloroperoxidase, galactose oxidase M3-5, periplasmic aldehyde oxidase, and lipase or a mixture of these enzymes. Nevertheless, other drawbacks for these approaches, such as the low concentration of substrates (around 2 to 100 mM) used for enzymatic reactions, accumulation of by-products and inefficient processes, impede practical applications. Whole cell conversions are more promising compared to enzymatic catalysis and have advantages such as high stability, low cost (no need for enzyme purification), cofactor regeneration, degradation of reactive oxygen species, and incorporation of multi-step reactions in one strain. In this review, I included all the methods from the researches that have been used recently, and I will discuss them one by one. In addition, it will be find out in which study the greatest yield of FDCA was achieved, the advantages of all methods will also be discussed.

Methods for biosynthesis from HMF to FDCA

Oxidation

The first research by Koopman, which was in 2010, he identified a novel Cupriavidus basilensis HMF / furfural oxidoreductase (HMFO), which converts HMF into FDCA. Pseudomonas putida S12 was inserted into the oxidoreductase system. 30 g/L of FDCA produced from HMF at a yield of 97per cent in fed-batch experiments using glycerol as the carbon source. It shows a very high yield of the product. This technique was copyrighted, as well. A commentary on the microbial metabolism of HMF and other furans was recently published by the same scientists.

The next research by Mara and co-worker (2013) stated that the peracids generated in situ that were formed in the presence of lipases as biocatalysts, could successfully oxidize DFF to FDCA. Using lipases as biocatalysts, alkyl esters as acyl donors, and hydrogen peroxide aqueous solutions (30% v / v) added step by step, peracids were produced in situ, which subsequently oxidized DFF to FDCA with high yield (gt; 99%) and excellent selectivity (100%). But, this process has been inactive for HMF. The use of DFF as a feedstock for FDCA synthesis involves a further step in the oxidation of HMF into DFF. Therefore, this method is very expensive for producing FDCA. 
In addition, a Caldariomyces fumago chloroperoxidase was found to have the biocatalytic ability against oxidation of HMF into FDCA. However, this process could not provide full HMF oxidation, providing FDCA yields of 60−75 percent and HFCA yields of 25−40 per cent. This property makes the C. Fumago chloroperoxidase a poor biocatalyst for the production of FDCA, particularly where very high purity FDCA is needed for specific applications such as polymer making. A fermentation cycle using Pseudomonas putida S12 to host oxidoreductase from Cupriavidus basilensis HMF for oxidation of HMF into FDCA was later studied. 30.1 g/L of FDCA was derived from HMF with a yield of 97 percent in fed-batch experiments using glycerol as the carbon source. Using acid precipitation and subsequent extraction of tetrahydrofuran, FDCA was recovered from the seed broth as a 99.4 per cent pure dry powder. This cycle is focused on both the oxidae and dehydrogenase activities. Most enzymes are restricted to either alcohol aldehyde oxidation, whereas full HMF oxidation to FDCA allows alcohol and aldehyde groups to act on the enzyme. In Fraaije and colleagues’ study (2014-2015), it was described a glucose-methanol-choline oxidoreductase (GMC) family of FAD-dependent oxidase, called HMF oxidase (HMFO), which showed high catalytic activity towards HMF Oxidation. Up to 95 per cent FDCA yield was achieved with complete HMF conversion at ambient pressure and temperature but requiring a long reaction time of 24 h at a low HMF concentration of 2 mM. Experiments have verified that HMF oxidation underwent two routes via this process (Figure 1).

Figure 1. Reaction routes of the enzymatic oxidation of HMF into FDCA by HMF oxidase

The step-wise bio-oxidation of HMF using two isolated oxidase variants, galactose oxidase M3–5 (GOaseM3–5) and periplasmic aldehyde oxidase ABC (PaoABC), with the addition of catalase to remove the formed hydrogen peroxide for both enzymes (Fig . 2a). The benefit of this method is that PaoABC catalyzes the complete double oxidation of DFF to FDCA, and does not involve the hydrated form of FFCA. Additionally, PaoABC uses the peroxygenase oxygen from air, rather than hydrogen peroxide. This led to a much higher conversion rate and in effect tolerated higher substratum concentration (gt; 100 mM). However, step by step addition of GOaseM_3-5 and PaoABC was required because the rate of GOaseM_3–5 HMF oxidation could not compete with PaoABC 's rapid oxidation of HMF to give HMFCA (see Fig . 1), itself a weak substratum for GOaseM_3–5, thus creating a bottleneck.

Figure 2. (a) Original stepwise and (b) new continuous one-pot conversion for HMF to FDCA using HRP

Scientists have chosen to look at PaoABC's reaction kinetics to decide whether they can control the reaction conditions to continuously supply FDCA and reduce HMFCA formation. For PaoABC with HMF as the substratum the apparent Km and Vmax values are 0.13 mM and 0.92 μmol/min*mg, respectively. Additionally, uncompetitive inhibition was observed with higher concentrations of HMF. It has long been established that horse radish peroxidase (HRP) activates GOase, and the addition of HRP to GOase catalyzed oxidation contributes significantly to increased activity and yield. Indeed, the addition of HRP increased the oxidation rate of HMF to enhance DFF conversion in a rapid 1-hour reaction (Table 1, cf. entries 1 amp; 2). Various pH signs suggested that pH 7.0 was the optimum for HMF oxidation (Table 1, entry 3). WT GOase was later stated to be performing well in unbuffered systems. This would reduce the costs associated with the scale up of this process; however, in unbuffered water, the M3–5 variant performed poorly (Table 1, Entry 5). Additionally, KPi has been shown to be a weak buffer for the wild form enzyme beforehand. It was postulated that when the copper-dependent GOase was incubated in phosphate buffer, a precipitate of Cu₃(PO₄)₂ could be produced, resulting in copper deficiency and lower activity. The M3–5 variant performed better in KPi, though, even at 100 mM 1 (Table 1, Entry 7). Strangely enough, it has developed that HMFCA is an exceptional substratum of GOaseM3–5, with and without HRP. The likely explanation for this is that HMFCA's GOaseM3–5 oxidation is partially inhibited by FDCA produced in the dual enzyme system. With improved conditions in hand for GOaseM3–5-HRP, scientists turned their attention to PaoABC. By changing the PaoABC concentration in the reaction it may reduced the rate of off-target oxidation of HMF and restrict the formation of HMFCA. It was pleased to note that it was able to generate the desired compound FDCA in a continuous rather than step-by - step manner at each concentration tested (Table 2, inputs 1–3). Analysis of the time course showed that HMFCA remained weak at all times at lower concentration of PaoABC (Table 2, Entry 1). Increasing quantities of HMFCA are produced as intermediates at higher PaoABC concentrations (entries 2 amp; 3), while full conversion to FDCA is still observed over the same time span (3 h) with HRP present. This confirms our initial finding that PaoABC would preferentially oxidise DFF in the presence of HMF and DFF. Incomplete conversion to FDCA (55%) was observed as the substrate HMF concentration increased to 100 mM and HMFCA 3 was present as 45 per cent of the reaction mixture (Table 2, Entry 4). Once again, reducing the amount of PaoABC decreased aldehyde group oxidation in HMF, resulting in lower conversion to HMFCA and 100per cent conversion to FDCA (Table 2, Input 5).

Table 1. Optimisation of HMF 1 to DFF 2 conversion by GOaseM3–5. A) Reaction conditions: Final volume 0.3 mL, 33 μL catalase (3.3 mg mL⁻¹), 70 μL HRP (1.0 mg mL⁻¹), 37 °C, 103 μL GOaseM3–5, (3 mg mL⁻¹), 1 h. Conversion calculated by RP-HPLC and adjusted with a 1:1:1 standard of HMF:DFF:FFCA. B) No HRP.

Table 2. Optimisation of PaoABC in the continuous oxidation of HMF to FDCA. Reaction conditions: Final volume 0.3 mL, 70 μl HRP(1 mg m⁻¹), 33 μL catalase (3.3 mg mL⁻¹), 0.2 mM KPi pH 7.0, 37 °C, 3 h, pH adjusted with 2 M NaOH.

While HMF could produce FDCA with almost quantitative yields, the HMF costs are high. Carbohydrates including fructose, glucose, and cellulose are slightly cheaper and more abundant than HMF. Hence, the oxidative conversion of carbohydrates into FDCA by one-pot reaction over multiple functional catalysts mixing acidic and metal sites is more desirable to conduct. The main problem with the direct conversion of carbohydrates into FDCA is the possibility of simultaneous carbohydrate oxidation. With the different example, in 2000, Kröger and colleagues realized the one-pot conversion of fructose to FDCA using the water / methyl isobutyl ketone (MIBK) two-phase device technique. As seen in Figure 3, the reaction was carried out with a polytetrafluorethylene membrane in a divided membrane reactor to prevent oxidation of the fructose.

Figure 3. Scheme of the processes in membrane reactor: 1 – HMF formation in water phase, 2 − diffusion of HMF in MIBK phase, and 3 − HMF oxidation

Fructose first dehydrated into HMF in water as a strong acid catalyst with a Lewatit SPC (trade name) 108. HMF was then removed to MIBK due to the higher solubility of HMF in MIBK, followed by oxidation into FDCA over metal catalysts, while fructose was insoluble in MIBK, thus preventing oxidization of fructose. This process provided FDCA's highest yield of 25 per cent. Interestingly, the authors noted that, in a pure MIBK step, HMF oxidation with this catalyst yielded DFF as the main product. The authors reported that water was required as a cosubstrate to form FDCA as the end product for oxidation of the aldehyde group.

Figure 4. Proposed oxidation pathway of HMF to FDCDM

Diffusion through the membrane affected the overall reaction rate. Additionally, levulinic acid (25% yield) has also been produced as the by-product. This method provided low FDCA yield, and the purification of FDCA from the byproducts was difficult. Later, Ribeiro and Schuchardt prepared a bifunctional catalyst by encapsulating Co(acac)3 in sol−gel silica, mixing acidic and redox power, and studied the one-pot conversion of fructose to FDCA, providing 72 per cent and 99 per cent FDCA selectivity for fructose conversion. Compared to the method stated by Kröger and coworkers, FDCA yield and selectivity have been improved by a multiple factor. Nevertheless, the reaction was conducted at 165 ° C and 20 bar high air pressure which is difficult to apply in practical application. Recently, a two-step process for converting fructose into FDCA has been documented by Zhang and colleagues. Fructose dehydration to HMF was achieved in HCl catalyzed isopropanol. Isopropanol was extracted by evaporation for next run after reaction. HMF was then extracted with water-extraction followed by oxidation over the catalyst Au / HT. Fructose has reached an average FDCA yield of 83 per cent. The step towards water extraction is very necessary for the entire process. Under the same conditions, the oxidation of HMF without extraction gave FDCA's yield only 51 per cent even after a long 20-hour cycle, although that was 98 per cent after 7 hours of water extraction. The authors reported that deactivation of the Au/HT catalyst was achieved by impurities such as humins. This method also provided an overall FDCA yield of 52 per cent using artichoke tuber from Jerusalem (major component of the fructose unit) as the feedstock. Similar work was later recorded by the same community, where in the first step polybenzyl ammonium chloride resins were used as a solid catalyst for fructose dehydration, providing FDCA with a fructose yield of 72 per cent. More recently, Zhang and colleagues have demonstrated a triphasic reactor capable of converting sugars to FDCA in a one-pot process. The triphasic mechanism consists of TEAB or water (phase I)−methyl isobutyl ketone (MIBK) (phase II)−water (phase III). The sugars (fructose or glucose) were first dehydrated to HMF in Phase I during the designed triphasic setup. HMF was then removed, purified, and transferred via a bridge to Phase III (Phase II). Eventually, in Phase III, HMF was oxidized to FDCA over the Au/HT catalyst. Overall, FDCA yields were obtained with fructose and glucose feedstock, respectively, of 78 percent and 50 percent. Phase II plays several roles: as a bridge for extracting, transporting and purifying HMF. To facilitate the recycling of the catalysts and to reduce the cost of the catalyst, our group has recently developed the one-pot conversion of fructose to FDCA through a two-step method by combining two magnetic catalysts. Generated from fructose dehydration over the DMSO catalyst Fe3O4@ SiO2−SO3H acid. Then, an external magnet quickly separated the magnetic Fe3O4@SiO2−SO3H from the reaction system, and HMF was then oxidized into FDCA with t-BuOOH over the nano-Fe3O4−CoOx catalyst in the remaining reaction solution. Based on the starting fructose, FDCA was obtained at a yield of 59.8 per cent after 15 h. Our developed method shows two distinct advantages : (1) the use of a magnetic catalyst facilitates the recycling of the catalyst; (2) the use of a transition-metal catalyst makes this method much more cost-effective for the practical synthesis of FDCA from renewable carbohydrates. Far more appealing is the direct conversion of carbohydrates into FDCA, but the latest findings are not satisfactory. For the development of FDCA the carbohydrate is dependent on fructose. The creation of novel catalysts with several catalytic sites for the conversion of other carbohydrates including glucose or even cellulose into FDCA should be compensated much more for. It is necessary to isolate several catalytic sites in order to avoid side reactions to realize a one-pot conversion of carbohydrates to FDCA.The acidic sites found in a hydrophilic environment will support the carbohydrate adsorption and facilitate carbohydrate dehydration into HMF and HMF release into the reaction solution. The oxidative sites found in a hydrophobic environment increase the absorption of the intermediate HMF and facilitate its oxidation to FDCA, as well as releasing the high polar product FDCA in the reaction solution simultaneously.

Whole-cell biocatalysis

HMFH and HMFO were respectively and co-expressed in R.ornithinolytica BF60 in the study in order to boost the development of FDCA from HMF via whole-cell biocatalysis.

R. Ornithinolytica BF60 was lab-insulated and marked. E. coli JM109 was used for the construction and replication of plasmids. At a final concentration of 100 mg /L (ampicillin), or 50 mg /L (kanamycin), antibiotics were added to the media. E. coli JM109 was cultured at 37 °C in Luria-Bertani medium (LB medium: 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) at 220 rpm in an Erlenmeyer flask.

R. ornithinolytica BF60 has cultured at 30 °C in minimal medium [MM medium, adapted from another study (Hartmans et al., 1989)] with glycerol as a carbon source. The medium was composed of the following ingredients (per liter): 0.5 g of yeast extract, 19.6 g of K2HPO4·3H2O, 10.6 g of NaH2PO4·2H2O, 2.0 g of (NH4)2 SO4, 0.4 g of MgCl2·6H2O, 4.0 mg of ZnSO4·7H2O, 2.0 mg of CaCl2·2H2O, 20.0 mg of EDTA, 10.0 mg of FeSO4·7H2O, 0.4 mg of Na2MoO4·2H2O, 0.8 mg of CuSO4·5H2O, 0.8 mg of CoCl2·6H2O, 2.0 mg of MnCl2·2H2O, and 10.0 g of glycerol.

R. ornithinolytica BF60 cells were incubated for 12 h at 30 ° C in 250 mL Erlenmeyer flasks containing 25 mL of MM medium with or without kanamycin for the wild or recombinant strains, respectively, for the preparation of the whole cell biocatalyst. Precultures were then inoculated into 2-L Erlenmeyer flasks with a Terrific-Broth size of 400 mL (TB form: 20 g / L of tryptone, 24 g / L of yeast extract, 4 mL / L of glycerol, 17 mM of KH2PO4 and 72 mM of K2HPO4) and 0.2 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) produced at 600 nm (OD600) of 0.6 optical density. After 24 h of incubation, the cells were extracted by centrifugation (8000 g for 20 min, 4 ° C), then washed three times with a 50 mM phosphate buffer (pH 8.0). In the same buffer the cell pellets were eventually resuspended to OD600 of 100. Biotransformation experiments were conducted in 100 mL shake flasks that contained 10 mL of a cell suspension and 100 mM HMF as a substrate. The test was carried out with 45.0 g / L whole-cell biocatalyst in a 50 mM phosphate buffer ( pH 8.0). To a concentration of 100 mM HMF has been added. The samples were incubated (20 mL) on a rotary shaker at 30 ° C, and the supernatant was collected by centrifugation at 14000 g for 10 min and passed via a 0.22-μm filter before inspection. The whole-cell biocatalytic activity (qFDCA) was measured at the time of reaction when each reaction reached its peak. HPLC has studied the development of FDCA, as described below.

In this analysis, the metabolic efficiency of R. ornithinolytica BF60 in MM medium was investigated at 20 mM HMF. Figures 5 and 6 show the cell growth time course as well as the wild-type and engineered R. ornithinolytica BF60 strains for HMF degradation. When R. ornithinolytica BF60 was grown in MM medium in the presence of 20 mM HMF (Figure 5) no apparent change in cell growth was noted. Nonetheless, the lower production of biomass by recombinant strains as opposed to the wild-type strain could be caused by the recombinant plasmid load, which includes an antibiotic resistance gene. Next, wild-type strain degradation of the HMF was studied (Figure 6A). The concentration decreased rapidly after HMF was applied, and HMF alcohol formation was observed within 12 h. Then, the HMF alcohol concentration slowly decreased, and the formation of HMF acid and FDCA began. HMF acid and FDCA accumulation was observed after 36 h and 60 h, respectively. A small amount of FFA, on the other hand, was detected, and no DFF accumulation was observed. Thus, R. ornithinolytica BF60 expressed the two enzymes, HMFH and HMFO, that can oxidize HMF alcohol to form FDCA. The resulting strains were known as ROBF60-H (HMFH expressing), ROBF60-O (HMFO expressing), and ROBF60-HO (HMFH and HMFO co-expressing).

In ROBF60-H cultures (Figure 6B), alcohol concentrations of HMF and HMF changed in a manner similar to the changes observed in the first 24 hour reaction with the wild-type strain.

The amount of HMF alcohol decreased rapidly, and that of FDCA increased rapidly. In the reaction mixture small amounts of FFA and HMF acid formed. Figure 6C shows the pattern of ROBF60-O strain HMF conversion; this pattern was identical to wild-type strain pattern. In addition, HMF accumulated after 12 h of complete conversion, then HMF and HMF alcohol quantities both decreased with FDCA, HMF acid, and FFA formation. The ROBF60-HO strain HMF biodegradation pattern (Figure 4D) was close to that of the ROBF60-H strain, but some aggregation of HMF and HMF acid was observed.

Thus, a full-cell biocatalyst for FDCA development was considered in the present analysis. In the previous study on wild-type R. ornithinolytica BF60 as a biocatalyst, 7.9 g/L (equivalent to 50.6 mM) FDCA was developed under optimum conditions at a molar conversion ratio of 51 per cent (45 g/L whole-cell biocatalyst, 100 mM HMF, 30 °C, and 50 mM phosphate buffer pH 8.0) (Hossain et al., 2017). In that bioconversion process, HMF alcohol and HMF acid were found to strongly accumulate, and the whole-cell biocatalytic activity (qFDCA) of the wild-type strain was 11.84 ± 0.05 μmol/([g CDW]·h). To create a more efficient biocatalyst for production of FDCA from HMF, the ROBF60-H, ROBF60-O, and ROBF60-HO strains were constructed here to be used as a whole-cell biocatalyst. Thus, when R. ornithinolytica BF60 expressed HmfH or HMFO, qFDCA increased to 25.08 ± 0.73 and 14.29 ± 0.07 μmol/([g CDW]·h), respectively. In addition, the mean ROBF60-H and ROBF60-O strain FDCA titer increased to 80.8 and 76.9 mM, respectively (Figure 7). After that, when HmfH and HMFO were co-expressed in R. ornithinolytica BF60 (ROBF60-HO) for complete conversion of the HMF acid and HMF alcohol to FDCA, final qFDCA increased to 27.32 ± 2.89 μmol/ ([g CDW]·h), and the FDCA production titer reached 93.6 mM, and the molar conversion ratio of HMF to FDCA reached 93.6 per cent.

Figure 5. The cell growth of different recombinant R. ornithinolytica BF60 strains in the presence of 20 mM HMF. Wild-type, harboring blank pBBR1MCS2 plasmid; ROBF60-H, expressing HmfH enzyme; ROBF60-O, expressing HMFO enzyme; ROBF60-HO, co-expressing HmfH and HMFO enzymes. The data shown are mean values from triplicates with error bars indicating the standard deviation.

Figure 6. The HMF degradation with different recombinant R. ornithinolytica BF60 strains in batch cultures with 20 mM HMF. (A) wild-type; (B) ROBF60-H; (C) ROBF60-O; (D) ROBF60-HO. Signal denotes: ■, FDCA; △, HMF acid; □, FFA; ▽, HMF; ♢, HMF alcohol. The data shown are mean values from triplicates with error bars indicating the standard deviation.

Figure 7. FDCA production in a whole-cell biotransformation from HMF. Wild-type, harboring blank pBBR1MCS2 plasmid; ROBF60-H, expressing HmfH enzyme; ROBF60-O, expressing HMFO enzyme; ROBF60-HO, co-expressing HmfH and HMFO enzymes. The data shown are mean values from triplicates with error bars indicating the standard deviation

In the biotransformation of HMF to FDCA, catalytically active and stable strains, Methylobacterium radiotolerans (G-2), and Burkholderia cepacia (H-2) were added. The isolated strain B. cepacia could produce FDCA of 1276 mg L⁻¹ from 2000 mg L⁻¹ of HMF under optimal conditions with 3 per cent cell biomass. An increase in  temperature speeds up the biotransformation process, when using the optimum temperature (28 °C) to convert HMF to FDCA. The isolated strain B. cepacia could produce FDCA of 1276 mg L⁻¹ from 2000 mg L⁻¹ of HMF under optimal conditions with 3 per cent cell biomass.  Increasing the HMF concentration may increase the FDCA yield but at higher concentration the recovery is inefficient. A lower concentration (lt; 1500 mg L−1) can promote limited cell growth while higher concentrations (gt; 2000 mg L−1) can decrease cell density due to higher concentration HMF toxicity. The FDCA created also contributes to a reduced pH, which is also unfavorable to cell production.
A HMF to FDCA fermentative synthesis process with the biocatalyst Pseudomonas putida S12 to promote oxidoreductase from Cupriavidus basilensis HMF14 was investigated using glycerol as a carbon source. As discussed earlier, the FDCA yield of 97per cent (from HMF feedstock) with 99.4per cent solid recovery from the crop broth was obtained. In the biotransformation of HMF to FDCA, catalytically active and stable strains, Methylobacterium radiotolerans (G-2), and Burkholderia cepacia (H-2) were added. The isolated strain B, under optimum conditions with 3 percent cell biomass. Cepacia could generate 1276 mg L−1 FDCA out of 2000 mg L−1 of HMF. An increase in temperature speeds up the rate of biotransformation, while the optimum temperature (28 °C) is used to convert HMF to FDCA. Increasing the HMF concentration may increase the FDCA yield but at higher concentration the recovery is inefficient. A lower concentration (lt; 1500 mg L−1) can promote limited cell growth while higher concentrations (gt; 2000 mg L−1) can decrease cell density due to higher concentration HMF toxicity. The FDCA created also contributes to a reduced pH, which is also unfavorable to cell production.

Microorganism isolation was conducted from soil samples obtained from 25 different Sikkim State locations in India, with altitudes ranging from 1120 to 4272 m above sea level. Isolating possible micro-organisms from 5-Hydroxymethyl furfural for the development of FDCA. The assembled soil samples were pre-cultured on minimal salt solution (MSS composition: g/l: MgSO4·7H2O, 0.2 g; CaCl2·2H2O, 0.002 g; KH2PO4, 0.5 g; K2HPO4, 0.5 g; NH4Cl, 0.5 g; Trace elements solution 10 mL/l, Trace element composition mg/l: FeSO4·7H2O, 300 mg; MnSO4·H2O, 50 mg; CoCl2·6H2O, 34 mg; Na2MoO4·2H2O, 34 mg; ZnSO4·7H2O, 40 mg; CuSO4·5H2O, 50 mg) with varying concentrations of HMF (500 mg/l–2500 mg/l) for 24 h. This was accompanied by spreading of precultured samples on four different media (MSS agar, nutrient agar, potato dextrose agar, and yеast extrаct peptone dextrose agar) with specific concentratiоns of HMF (500 mg / l–2500 mg / l) following normal serial dilution protocol. The plates were incubated at 28 ° C and 37 ° C for a maximum of 48 h. To obtain a single colony, morphologically distinct isolates were streaked to fresh plates. Human, pure cultures have been further screened for FDCA development. Acinetobacter oleivorans S27 were grown at 30 ° C for 12 h on a rotary shaker (200 rpm) in a medium (100 mL) of Luria- Bertani (LB) for pre-inoculum preparation. One percent (v / v) of the pre-inoculum was transferred to a freshly prepared LB medium (100 mL) and held for 27 h for incubation under the same conditions. Cells were harvested for 15 min after 27 h of incubation by centrifugation at 12,000 RPM at 4 ° C. Cells were washed twice with sterilized water and a phosphate buffer ( pH 8) to remove full media components. Then, the cell pellet was transferred to the mineral salt media (50 mL) as a sole source of carbon, supplemented with 500 mg / L HMF. The output media is held on a rotary shaker at 30 ° C (200 rpm) for further study. In every 24 hours, the supernatant was separated from the reaction mixture and analyzed using high-performance liquid chromatography (HPLC). 
For the FDCA development, more than 10 strains which grow in the presence of HMF were initially selected. The strain S27 (given product code) from the chosen strain list may grow in HMF to a concentration of up to 3000 mg / L, and demonstrates more FDCA manufacture than other strains. The organism was described as Acinetobacter Oleivorans after sequencing of the rRNA gene in 16s. Series with accession no. (MK359024) was deposited at GenBank. The S27 Acinetobacter oleivorans has been found to be a gram-negative and resistant to oxidase. It shows good activity on the output of FDCA when HMF provides as the sole source of energy. 
A best model of whole-cell biocatalyst should have some good features, such as genetically flexible, fast-growing capability in a simple medium, fair-quantity enzyme production, and downstream process compatibility with current approach system. The focus of this analysis was therefore primarily on the development of FDCA and the optimisation of biotransformation parameters. The optimization began with inoculum age because the time of the process of cell harvesting has a significant influence in the level of development. The majority of the enzymes involved in the biotransformation will be generated in a particular step. Moreover the late exponential period indicated by studies is the crucial time for growth. It was found that the average conversion was found to be best between 24 and 28 hours (Figure 8) and 27th hours. The single optimization of the incubation temperature parameters resulted in 30 ° C (Figure 9). Less activity was noticed as temperature rises up to 37 ° C and most likely this is occurring due to denaturation of the enzyme. Temperature increase does not make a difference and in the case of gram negative species Acinetobacter 30 ° C has also been identified as optimum temperature in the case of diesel oil degradation. Broad range of pH (6–8.5) studies were selected for pH optimization, and results showed optimum pH 7.5 (Figure 10). The pH tuning playеd an important role in the level of prоduction. The pH of the reaction mixture is acidic due to the other derivatives that include alcohol and acids. Additionally pH has been preserved during the time of incubation. The data suggests the efficient concentration of the 500 mg to 3000 mg substrates (Figure 11). The strain could use the HMF at a concentration of 90 per cent up to 2000 mg / L (data is not shown). FDCA yield was found to be approximately 51 per cent when the initial 500 mg / L HMF concentration was measured. Higher than 2000 mg/L of HMF concentration, because of toxicity to the organism, the reaction was inhibited.

Figure 8. Optimization of inoculum age

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