Bioconversion Yield Research Articles

Bioconversion of vitamin D3 to calcifediol by using resting cells of Pseudonocardia sp.

Resting cells of Pseudonocardia sp. KCTC 1029BP were used for the bioconversion of vitamin D3 to calcifediol which is widely used to treat osteomalacia and is industrially produced by chemical synthesis. To obtain the maximum bioconversion yield of calcifediol by the microbial conversion of vitamin D3, a two-step optimization process was used, including the Plackett-Burman and the central composite designs. Six variables, namely agitation speed, aeration rate, resting cell concentration, vitamin D3 concentration, temperature, and pH, were monitored. Of these, aeration rate, resting cell concentration, and temperature were selected as key variables for calcifediol production and were optimized using the central composite design. Optimal bioconversion conditions obtained were as follows: aeration rate of 0.2 vvm, resting cell concentration of 4.7% w/v, and temperature of 33 °C. Using the optimal conditions, 356 mg calcifediol l(-1) was obtained with a bioconversion yield of 59.4% in a 75 l fermentor. These are the highest values reported to date.

Combined steam-explosion toward vacuum and dilute-acid spraying of wheat straw. Impact of severity factor on enzymatic hydrolysis

This study deals with the development of an eco-friendly hydrothermal pretreatment of wheat straw. This pretreatment involves moderate temperatures without generation of liquid fractions allowing to reduce the energy input consumption and the environmental impact and enhance the enzymatic hydrolysis. For this purpose, the synergistic effect of impregnation of wheat straw by dilute sulfuric acid and DIC (in french: Détente instantannée Contrôlée) pretreatment on enzymatic hydrolysis has been investigated. DIC process involves subjecting the lignocellulosic biomass to saturated steam pressure (0.3–0.7 MPa), followed by a sudden decompression toward vacuum (5 kPa). The optimization of processing conditions was carried out using a full-factorial design, in respect to processing temperature (133–165 °C), residence time (5–40 min) and sulfuric acid concentration (0.70–2.20%). These processing conditions were converted into a single combined severity factor (CS), relating pH, temperature and residence time of pretreatment, ranged from −2.22 to 1.25. The efficiency of proposed pretreatment was measured through the enzymatic digestibility of lignocellulose and the responses parameters of experimental design were the monomeric glucose and xylose. The most influential factor on biomass bioconversion was sulfuric acid concentration followed by temperature and processing time. The optimum values were 2.2%, 165 °C and 40 min, for acid concentration, temperature and residence time, respectively. Strong correlations were observed between the enzymatic hydrolysis and conditions of pretreatment through the analysis of CS. The intense conditions have contributed to disrupt the biomass structure make it less dense leading to a higher specific surface area (ABET). Thus, increasing in accessibility of cellulose to enzymes has improved the rate and yields of bioconversion.

The effect of Pleurotus ostreatus arabinofuranosidase and its evolved variant in lignocellulosic biomasses conversion

The fungal arabinofuranosidase from Pleurotus ostreatus PoAbf recombinantly expressed in Pichia pastoris rPoAbf and its evolved variant rPoAbf F435Y/Y446F were tested for their effectiveness to enhance the enzymatic saccharification of three lignocellulosic biomasses, namely Arundo donax, corn cobs and brewer’s spent grains (BSG), after chemical or chemical–physical pretreatment. All the raw materials were subjected to an alkaline pretreatment by soaking in aqueous ammonia solution whilst the biomass from A. donax was also pretreated by steam explosion. The capability of the wild-type and mutant rPoAbf to increase the fermentable sugars recovery was assessed by using these enzymes in combination with different (hemi)cellulolytic activities. These enzymatic mixtures were either entirely of commercial origin or contained the cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli in substitution to the commercial counterparts. The addition of the arabinofuranosidases from P. ostreatus improved the hydrolytic efficiency of the commercial enzymatic cocktails on all the pretreated biomasses. The best results were obtained using the rPoAbf evolved variant and are represented by increases of the xylose recovery up to 56.4%. These data clearly highlight the important role of the accessory hemicellulolytic activities to optimize the xylan bioconversion yields.

Biochemical characterization of a thermostable l-arabinose isomerase from a thermoacidophilic bacterium, Alicyclobacillus hesperidum URH17-3-68

Abstract The rare monosaccharide d -tagatose is a low-calorie sugar-substituting sweetener, having 92% of relative sweetness but only 1/3 of energy content of sucrose. Industrial production of d -tagatose is carried out from d -galactose by l -arabinose isomerase ( l -AI). It is generally recognized that commercial l -AI for d -tagatose production requires two important enzymatic properties, thermostability and slightly acidic pH optimum. In this article, a thermostable l -AI was characterized from a novel thermoacidophilic bacterium, Alicyclobacillus hesperidum URH17-3-68, which showed promising thermostability and displayed a relatively wide pH spectrum. The araA gene encoding the Al. hesperidum l -AI was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and ion-exchange chromatography. The enzyme displayed maximal activity at 70 °C and pH 7.0, and showed more than 75% of maximal activity from pH 5.5 to 7.0. Cobalt ion was required as optimum metal cofactor for both activity simulation and thermostability improvement at high temperature. The enzyme retained 93% and 63% of initial activity after 4 and 16 h of incubation, respectively, at 75 °C in the presence of Co2+. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) for substrate d -galactose were measured to be 54.7 mM, 68.0 min−1, and 1.2 mM−1 min−1, respectively. The bioconversion yield of d -tagatose by the purified enzyme after 27 h at 70 °C reached 43% and 22%, from 50 and 200 mM of d -galactose, respectively. Due to the promising thermostability and high activity at slight acidic pH, the Al. hesperidum l -AI was appropriate for use as a new source of d -tagatose producing enzyme.

Modeling lipase-catalyzed interesterification of flaxseed oil and tricaprylin for the synthesis of structured lipids

Abstract The biosynthesis of structured lipids (SLs) in organic solvent media (OSM) was carried out by the interesterification of flaxseed oil (FO) TAGs and tricaprylin (TC) using Lipozyme TL-IM from Thermomyces lanuginosus . The bioconversion yield (BY, %) of medium-long-medium type SLs (MLM-SLs), including CLnC (C-caprylic and Ln-linolenic acids), CLaC (La-linoleic acid) and COC ( O -oleic acid), was monitored. Response surface methodology (RSM) was used to obtain significant models for the responses and to optimize the interesterification reaction, on the basis of a three level, five variable fractional factorial design (FFD) with center points. For the optimization of the interesterification reaction significant parameters, including reaction time ( R t ), reaction temperature ( T r ), TC to FO molar ratio ( M r ), enzyme concentration ( E c ), and agitation speed ( A s , 100–300 rpm), were considered. The optimal conditions generated for the maximum synthesis of CLnC, CLaC and COC, were found to be, 4.00–4.01 h for R t , 41.49–50.00 °C for T r , 1.5% for E c , 5.00–5.13 mol/mol for M r and 260–300 rpm for A s . Under these optimal conditions, the BY of CLnC, CLaC and COC were predicted to be 35.34–35.45, 4.09–4.19 and 8.44–8.53%, respectively.

Optimization of Lipase‐Catalyzed Interesterification of Flaxseed Oil and Tricaprylin Using Response Surface Methodology

AbstractThe biosynthesis of structured lipids (SL) in organic solvent media was carried out by the interesterification of flaxseed oil (FO) and tricaprylin (TC), using Novozym 435. The bioconversion yield (BY, %) of medium‐long‐medium type SL, including C‐caprylic and Ln‐linolenic acids (CLnC), La‐linoleic acid (CLaC) and O‐oleic acid (COC), was monitored. Response surface methodology was used to obtain significant models for the responses, on the basis of a five level, five variable central composite rotatable design. In the experimental preliminary trials significant reaction parameters, including reaction temperature (Tr), TC/FO molar ratio (Mr), enzyme concentration (Ec), reaction time (Rt) and initial water activity (aw), were considered for optimization. Significant models for CLnC, CLaC and COC were determined after regression analysis with backward elimination. The optimal conditions, generated for a maximum CLnC, CLaC and COC, were found to be 54.50–56.25 °C for Tr, 6.23–6.25 mol/mol for Mr, 2.68–3.13 % for Ec, 36.58–37.50 h for Rt and 0.15–0.33 for aw. Under these optimum conditions, the BY of CLnC, CLaC and COC was predicted to be 32.48–36.67, 3.26–3.38 and 5.79–6.16 %, respectively.

Functional characterization of the sucrose isomerase responsible for trehalulose production in plant-associated Pectobacterium species

Fifty-three plant-associated microorganisms were investigated for their ability to convert sucrose to its isomers. These microorganisms included one Dickeya zeae isolate and 7 Enterobacter, 3 Pantoea, and 43 Pectobacterium species. Eleven out of the 53 strains (21%) showed the ability to transform sucrose to isomaltulose and trehalulose. Among those, Pectobacterium carotovorum KKH 3-1 showed the highest bioconversion yield (97.4%) from sucrose to its isomers. In this strain, the addition of up to 14% sucrose in the medium enhanced sucrose isomerase (SIase) production. The SIase activity at 14% sucrose (47.6U/mgdcw) was about 3.6-fold higher than that of the negative control (13.3U/mgdcw at 0% sucrose). The gene encoding SIase, which is comprised a 1776bp open reading frame (ORF) encoding 591 amino acids, was cloned from P. carotovorum KKH 3-1 and expressed in Escherichia coli. The recombinant SIase (PCSI) was shown to have optimum activity at pH 6.0 and 40°C. The reaction temperature significantly affected the ratio of sucrose isomers produced by PCSI. The amount of trehalulose increased from 47.5% to 79.1% as temperature was lowered from 50°C to 30°C, implying that SIase activity can be controlled by reaction temperature.

Application of a Chitosan-Immobilized Talaromyces thermophilus Lipase to a Batch Biodiesel Production from Waste Frying Oils

Waste frying oil, which not only harms people's health but also causes environmental pollution, can be a good alternative to partially substitute petroleum diesel through transesterification reaction. This oil contained 8.8 % of free fatty acids, which cause a problem in a base-catalyzed process. In this study, synthesis of biodiesel was efficiently catalyzed by the covalently immobilized Talaromyces thermophilus lipase and allowed bioconversion yield up to 92 % after 24 h of reaction time. The optimal molar ratio was four to six parts of methanol to one part of oil with a biocatalyst loaded of 25 wt.% of oil. Further, experiments revealed that T. thermophilus lipase, immobilized by a multipoint covalent liaison onto activated chitosan via a short spacer (glutaraldehyde), was sufficiently tolerant to methanol. In fact, using the stepwise addition of methanol, no significant difference was observed from the one-step whole addition at the start of reaction. The batch biodiesel synthesis was performed in a fixed bed reactor with a lipase loaded of 10 g. The bioconversion yield of 98 % was attained after a 5-h reaction time. The bioreactor was operated successfully for almost 150 h without any changes in the initial conversion yield. Most of the chemical and physical properties of the produced biodiesel meet the European and USA standard specifications of biodiesel fuels.

Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-β-xylosyl-10-deacetyltaxol and Its Analogues

Paclitaxel, a natural antitumor compound, is produced by yew trees at very low concentrations, causing a worldwide shortage of this important anticancer medicine. These plants also produce significant amounts of 7-β-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related β-xylosidases of these organisms have not yet been defined. We set out to discover an efficient enzyme for 10-deacetyltaxol production. By combining the de novo sequencing of β-xylosidase isolated from Lentinula edodes with RT-PCR and the rapid amplification of cDNA ends, we cloned two cDNA variants, Lxyl-p1-1 and Lxyl-p1-2, which were previously unknown at the gene and protein levels. Both variants encode a specific bifunctional β-d-xylosidase/β-d-glucosidase with an identical ORF length of 2412 bp (97% identity). The enzymes were characterized, and their 3.6-kb genomic DNAs (G-Lxyl-p1-1, G-Lxyl-p1-2), each harboring 18 introns, were also obtained. Putative substrate binding motifs, the catalytic nucleophile, the catalytic acid/base, and potential N-glycosylation sites of the enzymes were predicted. Kinetic analysis of both enzymes showed kcat/Km of up to 1.07 s(-1)mm(-1) against 7-β-xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with a conversion rate of over 85% and a highest yield of 8.42 mg/ml within 24 h, which is much higher than those reported previously. Therefore, our discovery might lead to significant progress in the development of new 7-β-xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7-β-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7-β-xylosyltaxanes and contribute to the growing database of glycoside hydrolases.

A comparison between immobilized pyrimidine nucleoside phosphorylase from Bacillus subtilis and thymidine phosphorylase from Escherichia coli in the synthesis of 5-substituted pyrimidine 2′-deoxyribonucleosides

Abstract Pyrimidine nucleoside phosphorylase from Bacillus subtilis ( Bs PyNP, E.C. 2.4.2.3) and thymidine phosphorylase from Escherichia coli ( Ec TP, E.C. 2.4.2.4) were used, as immobilized enzymes, in the synthesis of 5-halogenated pyrimidine 2′-deoxyribonucleosides ( 14 – 18 ) by transglycosylation in fully aqueous medium. From the comparative study of the two biocatalysts, no remarkable differences emerged about their substrate specificity, bioconversion yield, stability in organic cosolvents (DMF and MeCN). Moreover, both biocatalysts could be recycled for at least 5 times with no loss of the productivity. Both enzymes do not accept arabinonucleosides and 2′,3′-dideoxynucleosides as substrates, whereas they catalyze bioconversions involving 5′-deoxyribonucleosides and 5-halogenated uracils. The synthesis of compounds 14 – 18 proceeded at a similar conversion (33–68% for Bs PyNP and 25–62% for Ec TP, respectively). Immobilization was found to exert, for both the biocatalysts, a dramatic enhancement of stability upon incubation in MeCN. Optimization of 5-fluoro-2′-deoxyuridine ( 14 ) synthesis (pH 7.5, 10 mM phosphate buffer, nucleoside/nucleobase 3:1 molar ratio) and subsequent scale-up afforded the target compound in 73% ( Ec TP) or 76% ( Bs PyNP) conversion (about 9 g/L).

Lipase-catalyzed synthesis and characterization of flaxseed oil-based structured lipids

The biosynthesis of structured lipids (SLs) was carried out by the interesterification of flaxseed oil (FO) and tricaprylin (TC) in an organic solvent medium (OSM), using selected commercial lipases, including Amano DF, Novozym 435, Lipozyme TL-IM and Lipozyme RM-IM. The fatty acyl chains of the synthesized triacylglycerols (TAGs) were identified by atmospheric pressure chemical ionization/mass spectrometric (APCI/MS) analysis, while the fatty acid positional distribution of the MLM- and MML-SLs (M-medium and L-long chain fatty acids) was determined by silver-ion high-performance liquid chromatographic (Ag+/HPLC) analysis. The effects of reaction temperature (Tr, 30–50°C), enzyme concentration (Ec, 0.5–4%, w/v), initial water activity (aw, 0.05–0.43) and reaction time (Rt, 0–72h) on the efficiency of the enzymes, were studied. The bioconversion yield (%) of the synthesized MLM- and MML-SLs was monitored under the established reaction parameters for each lipase. The maximum yield of MLM-SLs was obtained in the order, of Novozym 435>Lipozyme TL-IM>Lipozyme RM-IM>Amano DF. Moreover, considering the ratio of the MLM- to MML-SLs produced by each enzyme, Novozym 435 and Lipozyme TL-IM were selected as the most effective enzymes for interesterification of FO and TC.

Selection method of pH conditions to establish Pseudomonas taetrolens physiological states and lactobionic acid production

Microbial physiological responses resulting from inappropriate bioprocessing conditions may have a marked impact on process performance within any fermentation system. The influence of different pH-control strategies on physiological status, microbial growth and lactobionic acid production from whey by Pseudomonas taetrolens during bioreactor cultivations has been investigated for the first time in this work. Both cellular behaviour and bioconversion efficiency from P. taetrolens were found to be negatively influenced by pH-control modes carried out at values lower than 6.0 and higher than 7.0. Production schemes were also influenced by the operational pH employed, with asynchronous production from damaged and metabolically active subpopulations at pH values lower than 6.0. Moreover, P. taetrolens showed reduced cellular proliferation and a subsequent delay in the onset of the production phase under acidic conditions (pH < 6.0). Unlike cultivations performed at 6.5, both pH-shift and pH-stat cultivation strategies performed at pH values lower than 6.0 resulted in decreased lactobionic acid production. Whereas the cellular response showed a stress-induced physiological response under acidic conditions, healthy functional cells were predominant at medium operational pH values (6.5-7.0). P. taetrolens thus displayed a robust physiological status at initial pH value of 6.5, resulting in an enhanced bioconversion yield and lactobionic acid productivity (7- and 4-fold higher compared to those attained at initial pH values of 4.5 and 5.0, respectively). These results have shown that pH-control modes strongly affected both the physiological response of cells and the biological performance of P. taetrolens, providing key information for bio-production of lactobionic acid on an industrial scale.

Lipase‐Catalyzed Synthesis of Medium‐Long‐Medium Type Structured Lipids Using Tricaprylin and Trilinolenin as Substrate Models

AbstractThe synthesis of medium‐long‐medium type structured lipids (SL) by the interesterification of tricaprylin (TC) and trilinolenin (TLN), using selected commercial lipases from Rhizomucor miehei (Lipozyme RM IM) and Candida antarctica (Novozym 435) was investigated. Although the bioconversion yield (BY) for Lipozyme RM IM (24.7 %) was close to that for Novozym 435 (24.0 %), the initial enzyme activity was 6.3 μmol CLnC/g enzyme/min and 1.6 μmol CLnC/g enzyme/min, respectively. Lipozyme RM IM was subsequently selected for further investigation. The structural analyses of SL indicated that the major products were 1,3‐dicapryl‐2‐linolenyl glycerol (CLnC) and 1(3)‐capryl‐2,3(1)‐dilinolenyl glycerol (CLnLn). In order to optimize the BY, selected parameters were investigated. The experimental results showed that using hexane as the reaction medium, at an initial water activity (aw) of 0.06, 10 mg solid enzyme/mL, substrate molar ratio of TC to TLN of 6:1 and a reaction time of 9 h, resulted in the highest BY (73.2 %). Using the optimized conditions, the effects of TLN concentration and other selective parameters, including the denaturation of the enzyme, controlling the aw and the addition of silica gel, on the mass productivity (PM), enzymatic productivity (PE) and volumetric productivity (PV) of the interesterification reaction, were also investigated.

α-Glucosidase 저해제 생산 균주, Gluconobacter oxydans CK-2165의 특성 및 배양 최적화

본 논문은 당뇨병 치료제로 알려진 Miglitol은 <TEX>${\alpha}$</TEX>-glucosidase 저해제로, 산업적으로 포도당과 에탄올아민으로부터 세 단계의 화학 및 생물전환 과정을 거쳐 합성되며. acetic acid bacteria에 속하는 Gluconobactor oxydans (G. oxydans)는 불완전 산화를 통해 1-deoxy-1-(2-hydroxyethylamino)-D-glucitol (P1)을 Miglitol의 전구체인 6-(2-hydroxyetyl) amino-6-deoxy-<TEX>${\alpha}$</TEX>-L-sorbofuranose (P2)로 생물 전환시키는 균주이다. 본 연구에서는 토양으로부터 스크리닝하여 선발된 고효율 생물전환 박테리아인 CK-2165의 균주를 동정하고 최적의 발효조건을 탐색하고자 하였다. 16S rDNA 서열과 계통수 분석결과 CK-2165는 G. oxydans에 속하는 미생물임을 결정하였으며, API 20E kits를 사용하여 선발된 균주의 탄소원 이용성을 실험한 결과 glucose, mannose, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, arabinose와 같은 탄소원에 대한 이용성을 가진 균주임을 확인하였다. 또한 산업적 생산을 위하여 배양 조건을 최적화하였고 균체를 이용하여 생물전환 반응에 중요한 요소들을 조사하였다. 생물전환 반응을 위해 사용되는 균체는 균 성장 단계 중 후기 정지기 (late-stationary phase)에 수확한 균체가 가장 높은 활성을 나타내었고 생물전환 반응에는 <TEX>$MgSO_4$</TEX>가 필수적임을 확인하였다. Miglitol, a well-known therapeutic intervention agents for diabetes, exhibits competitive inhibitory activity against <TEX>${\alpha}$</TEX>-glucosidase and it is usually produced through three sequential steps including chemical and bioconversion processes. Gluconobactor oxydans (G. oxydans) belonging to acetic acid bacteria biologically, converts 1-deoxy-1-(2-hydroxyethylamino)-D-glucitol (P1) into a key intermidiate, 6-(2-hydroxyetyl) amino-6-deoxy-<TEX>${\alpha}$</TEX>-L-sorbofuranose (P2) by incomplete oxidation. In this study, we identified and optimized fermentation conditions of CK-2165, that was selected in soil samples by comparing the bioconversion yield. CK-2165 strain was found to be closely related to G. oxydans based on the result of phylogenetic analysis using 16S rDNA sequence. Utilization of API 20 kits revealed that this strain could use glucose, mannose, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose as carbon sources. The culture conditions were optimized for industrial production and several important factors affecting bioconversion rate were also tested using mycelial cake. Cell harvested at the late-stationary phase showed the highest bioconversion yield and <TEX>$MgSO_4$</TEX> was critically required for the catalytic activity.

Methods for microbial screening and production of polyol oils from soybean oil through bioprocess

Abstract Soypolyol oils (oxygenated acylglycerols) are important starting materials for the manufacture of polymers such as polyurethane. Currently, they are produced by a two-step chemical process involving epoxidation and the subsequent opening of the oxirane ring. The objective of this study is to develop a bioprocess to produce polyol oils directly from soybean oil. For product separation, we found that TLC with a two step development solvent systems could separate the polyol products from substrate soybean oil. The products and substrates were separated in the following order: substrate triacylglycerols (Rf 0.8), free fatty acids (FA, Rf 0.7), product dihydroxy TAG (Rf 0.4), trihydroxy TAG (Rf 0.3), monohydroxy FA (Rf 0.1) and dihydroxy FA (Rf 0.05). We also found that HPLC with a C18 reverse phase column and a linear gradient of 100% methanol to 100% 2-propanol over 60 min at 1 mL/min flow rate was able to separate product polyol oils and substrate soybean oil. Free FA and polyol oils (diacylglycerols (DAG) containing hydroxyl FA) were eluted between 5 min and 15 min. DAG containing two normal FA was eluted between 15 min and 28 min and the substrate soybean oil was eluted between 36 min and 45 min. A total of 400 microbial cultures were screened and we identified 25 hits. Polyol oils' products were purified through a silica gel column chromatography, fractionated by HPLC and then analyzed by MS. A total of 57 molecular species of DAG containing tri-, di-, monohydroxy FA and normal FA were identified by MS. HPLC chromatogram of evaporative light scattering detector was used for semi-quantification of these DAG in the purified polyol oils. The total content of the DAG containing two normal FA was about 25% and the total content of DAG containing hydroxy FA might be about 75%. The yield of bioconversion by culture A01-35 from soybean oil to polyol oil products (DAG containing hydroxyl FA) plus DAG containing normal FA was 31% by weight. Bioconversion of soybean oil to polyol oils is a new research area without available methodology to follow. Here we report a new bioprocess for the production of soypolyol oils directly from soybean oil.

Ultrasonic and high-temperature pretreatment, enzymatic hydrolysis and fermentation of lignocellulosic sweet sorghum to bio-ethanol

This study investigated the conversion of lignocellulosic biomass to hexose and pentose sugars through ultrasonic and hot water pretreatment, followed by hydrolysis with a mixture of enzymes and fermentation by Saccharomyces cerevisiae to bio-ethanol. Lignin concentration decreased by 52%, cellulose and hemicellulose concentrations increased by 49% and 25%, respectively, after the ultrasonic plus hot water pretreatment. Cellulose conversion to glucose was the highest when Accellerase 1500 + XC enzyme was used (89%), followed by Accellerase 1500 + BG (84%), Accellerase 1500 + XY (83%) and Accellerase 1500 (82%). Hemicellulose conversion to xylose and arabinose was the highest when Accellerase 1500 + XC enzyme was used (48%), followed by Accellerase 1500 + XY (40%). Optimum yield of bioconversion of the cellulose was 3.2–4.2 g ethanol/100 g of sweet sorghum biomass and ethanol concentration was 8–10 g/L based on cellulose conversions. The results in this study can be used to optimise processing conditions for bioethanol production from lignocellulose.

Enzymatic synthesis of phenolic lipids in solvent-free medium using flaxseed oil and 3,4-dihydroxyphenyl acetic acid

The enzymatic synthesis of phenolic lipids (PLs) by transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA) was investigated in solvent-free medium (SFM), using Novozym 435 from Candida antarctica as the biocatalyst. The effects of selected reaction parameters, water activity (aw), enzyme concentration and agitation speed, were studied and optimized. Increasing the aw of the reaction mixture from 0.18 to 0.38 resulted in a significant increase in the bioconversion yield from 62 to 77%. APCI–MS analysis confirmed the formation of six 3,4-dihydroxyphenyl acetoylated lipids, which were monolinolenyl, dioleyl, dilinolenyl, linoleyl linolenyl, oleyl linolenyl and oleyl linoleyl dihydroxyphenyl acetates. The highest enzymatic activity (178nmol of PLs/g solid enzyme/min) was obtained using 40mg of solid enzyme (400PLU)/mL at agitation speed 150rpm. Using the optimized conditions, the phenolic lipids showed a high relative proportion of linolenic acid (C18:3n−3) that increased from 57% in the flaxseed oil to 75 and 64% in the produced phenolic mono- and diacylglycerols, respectively. In addition, the synthesized phenolic lipids demonstrated a 7.2-fold lower radical scavenging activity than that of DHPA but half that of α-tocopherol.

Lipase-catalyzed transesterification of krill oil and 3,4-dihydroxyphenyl acetic acid in solvent-free medium using response surface methodology

The optimization of the enzymatic transesterification in solvent-free medium (SFM) of krill oil with selected phenolic acids (PAs), using two immobilized lipases, was investigated. The use of Novozym 435 with 3,4-dihydroxyphenylacetic acid resulted in the highest bioconversion yield (BY). The central composite rotatable design was used to evaluate the effects of the PA concentration (PAC) and lipase concentration (LC) as well as the agitation speed (AS) on the BY of phenolic lipids. The initial findings indicated that the stationary point was a saddle point. To overcome this saddle point, sequential experiments have been carried out at fixed PAC, with LC and AS as the two independent variables. For the models with PAC, fixed at 10 and 20 mM, the results revealed that LC had a significant quadratic effect (P < 0.05) on the %BY, whereas a significant linear effect was only obtained at PAC fixed at 20 mM. The AS had a significant quadratic effect (P < 0.05) on the %BY only for the model with PAC fixed at 10 mM. At fixed PAC of 20 mM, the response surface model predicted a BY of 75%, using a LC of 62 mg/mL and an AS of 154 rpm. The subsequent verification experiment carried out under these conditions confirmed the validity of the prediction.

Lipase-catalyzed synthesis of structured phenolic lipids in solvent-free system using flaxseed oil and selected phenolic acids as substrates

Structured phenolic lipids (PLs) were obtained by lipase-catalyzed transesterification of flaxseed oil, in a solvent-free system (SFS), with selected phenolic acids, including hydroxylated and/or methoxylated derivatives of cinnamic, phenyl acetic and benzoic acids. A bioconversion yield of 65% was obtained for the transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA). However, the effect of the chemical structure of phenolic acids on the transesterification of flaxseed oil in SFS was of less magnitude as compared to that in organic solvent system (OSS). Using DHPA, the APCI-MS analysis confirmed the synthesis of monolinolenyl, dilinolenyl, linoleyl linolenyl and oleyl linolenyl dihydroxyphenyl acetates as phenolic lipids. A significant increase in the enzymatic activity from 200 to 270 nmol of PLs/g solid enzyme/min was obtained upon the addition of the non-ionic surfactant Span 65. However, upon the addition of the anionic surfactant, sodium bis-2-ethylhexyl sulfosuccinate (AOT), and the cationic one, hexadecyltrimethylammonium bromide (CTAB), the enzymatic activity was decreased slightly from 200 to 192 and 190 nmol of PLs/g solid enzyme/min, respectively. The results also showed that the increase in DHPA concentration from 20 to 60 mM resulted in a significant increase in the volumetric productivity (P(V)) from 1.61 to 4.74 mg PLs per mL reaction mixture per day.

Decrement of cellulose recalcitrance by treatment with ionic liquid (1‐ethyl‐3‐methylimidazolium acetate) as a strategy to enhance enzymatic hydrolysis

AbstractBACKGROUND: The high crystallinity of cellulose underlies the recalcitrance that this polymer presents in enzymatic degradation. Thus, a pre‐treatment step is applied in most bioconversion processes. Treatments with ionic liquids are considered an emerging pre‐treatment technology, owing to their high efficiency in solvating cellulose, over molecular solvent systems.RESULTS: Crystalline cellulose with and without ionic liquid (1‐ethyl‐3‐methylimidazolium acetate) treatment, both commercially available, were used as substrates in enzymatic hydrolysis reactions using the earlier evaluated cellulolytic system of Fusarium oxysporum. The in situ removal of the hydrolysate during reactions enhanced the reaction rate as well as the overall glucose production. Ionic liquid treatment significantly decreased cellulose crystallinity and enhanced bioconversion yields and rates. The effects of cellulose structural changes during treatment on hydrolysis rate were investigated and the recalcitrance constants were determined.CONCLUSION: The study showed that ionic liquid‐treated cellulose became more homogeneous and more easily degradable than the untreated cellulose, a conclusion that was expressed mathematically by the difference in the recalcitrance constants for the two substrates. It was concluded that glucose production from ionic liquid‐treated cellulose could achieve very high conversion yields in consolidated bioprocesses or during simultaneous saccharification and fermentation. Copyright © 2012 Society of Chemical Industry