Separation and partial purification of polysaccharides from fungi or mushrooms are difficult and have become the main bottleneck in the development of a commercially viable process. In this study, an aqueous two-phase system (ATPS) composed of short chain alcohols and salts was developed for the separation and partial purification of polysaccharides and other bioactive constituents from Cordyceps sinensis (CS). The results showed that ATPS not only purified CS polysaccharides (CSPS) with the removal of 99.1% cell debris, 80% proteins, 90.7% pigments, and 61.3% nucleic acids, but also simultaneously recovered CSPS, adenosine, and mannitol up to 96.6%, 100%, and 88.4%, respectively. Compared with traditional purification methods, the coupling of ethanol/Na2CO3 ATPS with ultrafiltration decreased the protein, nucleic acid, and pigment contents in the purified CSPS by 344%, 192%, and 290%, respectively. Therefore, this environmentally friendly method could have potential applications in large-scale separation of multiple bioactive constituents from fungi in the future.

Highly efficient interfacial enzymatic hydrolysis of oil was achieved in a three-liquid-phase system, wherein the substrate constituted one of the phases. The enlarged interfacial area and relieved product inhibition were responsible for the high catalytic efficiency. Convenient product isolation and the high reusability of the enzyme were also demonstrated.

A water-in-ionic liquid microemulsion ([Bmim]PF6/Tween20/H2O) was applied as reusable reaction medium to esterify phytosterols with fatty acid by Candida rugosa lipase (CRL) successfully. Two kinds of commercial CRLs, AY30 and AYS which cannot effectively catalyze esterification in conventional reaction system were found effective in the microemulsion system. Effects of reaction parameters on esterification were investigated; results showed that the conversion rate of 87.9 and 95.1 % was obtained in 24 and 48 h of reaction, respectively, under the optimized condition: the molar ratio of water to Tween 20 (w0 value) at 5.4, Tween 20 at a concentration of 305 mM, 50 °C,pH 7.4, 10 % of enzyme loading (w/w, with respect to total reactants), and phytosterols/lauric acid molar ratio of 1:2. Moreover, by using n-hexane as the extraction agent, the lipase-encapsulated microemulsion could be reused at least seven times (>168 h) without significant changes in the conversion rate, while achieving a purpose of simple separation and purification.

•Bioflocculants were produced from different culture medium with or without metal ion.•Three bioflocculants, PSB1-1, PSB2-1 and PSB3-1 were purified and characterized.•PSB1-1 exhibited the best performance in wastewater treatment of different pH.•A bioflocculant with high flocculating activity in the wide range of pH 3–9 was obtained.The production and purification of polysaccharide-based bioflocculants (PSBs) by Paenibacillus mucilaginosus GIM1.16 in metal ion-supplemented medium and basal medium were evaluated. Three purified PSB1-1, PSB2-1 and PSB3-1 possessed different monosaccharide composition and their molecular weights were 2.53 × 106, 7.77 × 106 and 13.2 × 106 Da, respectively. FT-IR spectrometry indicated the presence of hydroxyl, carboxyl and phosphate groups in the three samples. Scanning electron microscopy showed that they had linear structure. The potential of these PSBs on wastewater treatment was evaluated. Among them, PSB1-1 exhibited the best performance, as it had high flocculating activities (above 94%) at 0.5–4 mg/L and could achieve high flocculating activities (above 97%) in the kaolin suspensions of pH 3–9. PSB1-1 was the key factor that might explain the enhanced flocculating activity of the supernatant from metal ion-supplemented medium. The performance of PSB1-1 on industrial wastewater was also satisfactory. PSB1-1 might be a good candidate as bioflocculant.

Pure diacylglycerol (DAG) is of vital importance for the biomedical and dietetic properties research of DAG. In this study, we aimed to develop an effective process to produce DAG-mixture of regioisomers with high purity. Firstly, DAGs and monoacylglycerols (MAGs) were synthesized by enzymatic esterification of glycerol and free fatty acids (FFAs) from camellia oil with catalysis of Penicillium camembertii lipase, and the obtained reaction mixture was composed of 49.9 % DAG [33.4 % for 1,3-DAG and 16.5 % for 1,2 (2,3)-DAG], 31.6 % MAG and 18.5 % FFA. Secondly, a monoacylglycerol lipase (lipase CBD-MGLP), which was produced by recombinant Escherichia coli in our laboratory, was employed to hydrolyze MAG in the above reaction mixture, and the MAG content decreased to 1.9 % under the optimal conditions with 375 U/g (U/w, with respect to the mass of MAG in the mixture) of CBD-MGLP loading, temperature of 45 °C, mass ratio of esterification mixture to Tris–HCl buffer (w/w) 10:10, and pH of Tris–HCl buffer 9.0. Then, the hydrolytic products were further purified by molecular distillation at low temperature of 130 °C under a pressure of 10 Pa [equivalent to 377 °C at 101.325 kPa (1 atm)], and the DAG purity was up to 98.0 % (66.6 % for 1,3-DAG and 31.4 % for 1,2-DAG) in the final products. This indicated that two-step enzymatic reactions combined with molecular distillation at low temperature could be a feasible and prospective process to produce DAG-mixture of regioisomers with high purity.

Typoselectivity of crude CBD-T1 lipase (Geobacillus sp. T1 lipase fused with a cellulose binding domain) was investigated. Multi-competitive reaction mixtures including a set of n-chain fatty acids (C8:0, C10:0, C12:0, C14:0, C18:1 n-9, C18:2 n-6 and C18:3 n-3) and tripalmitin-enriched triacylglycerols were studied in hexane. The crude CBD-T1 lipase discriminated strongly against C18:1 n-9 [competitive factor (α) = 0.23] and showed the highest preference for C8:0 (α = 1). Utilizing the catalytic properties of crude CBD-T1 lipase, acidolysis of soybean oil with C8:0 was selected as a model reaction to investigate the ability of the lipase to produce MLM-type (medium-long-medium) structured lipids. Several reaction parameters (added water amount, reaction temperature, substrate molar ratio and reaction time) examined for incorporating C8:0 into soybean oil, the optimum conditions were: 1:3 (soybean oil/C8:0) of molar ratio, 3 mL of hexane, 50 °C of temperature, 48 h of reaction time, 20 % of crude CBD-T1 lipase (w/w total substrates), and 7.5 % of water (w/w enzyme). Under these conditions, the incorporation of C8:0 was 29.6 mol%. The results suggest that crude CBD-T1 lipase, which showed different fatty acid specificity profiles, is a potential biocatalyst for the modification of fats and oils.

A camellia oil-based isopropyl ester (CO-IPE) was successfully synthesized by enzymatic alcoholysis with camellia oil (CO), and its physiochemical properties were analyzed. Three commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM and Novozym 435) were screened, and Novozym 435 was the best one. The optimal reaction conditions were achieved at 240 U/g of Novozym 435 loading, a substrate molar ratio of 5:1 (isopropanol/CO), and 24 h of reaction time at 55 °C. Under the above conditions, the content of CO-IPE was obtained as 89.83%. Purity of CO-IPE further increased to be 96.95% after separation by rotary evaporation and molecular distillation. The viscosity of the synthesized CO-IPE showed itself to be about six times lower than that of CO, and the refractive index of the CO-IPE (1.449) was nearer to 1 in contrast to that of CO. It suggested that CO-IPE could be more intensively applied in the cosmetic industry.

Diacylglycerols (DAG) of conjugated linoleic acid (CLA) were prepared by esterification of glycerol with fatty acids enriched with CLA (FFA–CLA, >95%) in the presence of a novel lipase from Malassezia globosa (SMG1). Lipase SMG1 is strictly specific to mono- and diacylglycerols but not triacylglycerols, which is similar to the properties of lipase from Penicillium camembertii (lipase G 50), but lipase SMG1 showed preference on the production of DAG with the reaction proceeding. Low temperature was beneficial for the conversion of FFA–CLA into acylglycerols, the degree of esterification reached 93.0% when the temperature was 5 °C. The maximum DAG content (53.4%) was achieved at 25 °C. The rate of DAG synthesis increased as the enzyme loading increased. However, at lipase amounts above 240 U/g mixtures, no significant increases in DAG concentration were observed. The molar ratio of FFA–CLA to glycerol and initial water content were optimized to be 1:3 (mol/mol) and 3%. Lipase SMG1 showed no regioselectivity because the contents of 1,3-DAG and 1,2-DAG were 43.1% and 21.2% based on total content of acylglycerols. By calculating the ratio of 9c, 11t-CLA to 10t, 12c-CLA, it was indicated that lipase SMG1 showed a little preference to 10t, 12c-CLA at the sn-1(3) position of monoacylglycerols (MAG), while no selectivity for 9c, 11t-CLA at the sn-2 position of DAG was obviously found.

In order to search for valuable and extremely thermo-stable enzymes that could be used in the protein hydrolysis industry, the gene corresponding to a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2 (GtLAP) was cloned and expressed in E. coli. The recombinant enzyme was purified, and its characteristics were examined. Meanwhile, potential applications of GtLAP in the hydrolysis of anchovy proteins were also investigated. GtLAP was overexpressed in IPTG-induced E. coli BL21 (pET28a-LAP) as a soluble protein, and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 125 ± 8.75 U/mg proteins. The molecular mass of GtLAP was estimated to be 55 kDa by SDS-PAGE analysis. The optimal reaction temperature and pH of GtLAP were 70 °C and 8.0, respectively. Under optimal conditions, GtLAP showed a marked preference for Leu-p-nitroanilide, followed by Met- and Phe-derivatives. Activity of GtLAP was strongly stimulated by Ni2+ ions, but was strongly inhibited by Hg2+. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of GtLAP to various extents and further induce changes in enzymatic activity. Results of hydrolytic experiment showed that combining GtLAP with endogenous enzymes could significantly increase the degree of hydrolysis to anchovy proteins and concentrations of free amino acids in hydrolysates. In this regard, GtLAP could potentially be used in the protein hydrolysis industry.

1,3-Dioleoyl-2-palmitoylglycerol, an important triacylglycerol in infant formulas, was effectively enriched by a two-step process: (a) dry fractionation of leaf lard and (b) enzymatic acidolysis of the fractionated leaf lard. In step a, the 1,3-dioleoyl-2-palmitoylglycerol content was increased from 16.77 to 30.73% after programmed temperature treatment of the leaf lard at 60 °C for 20 min followed by 34 °C for 10 h. In step b, 43.72% of the 1,3-dioleoyl-2-palmitoylglycerol content was obtained at the optimal conditions of enzymatic acidolysis: a substrate molar ratio of 1:4 (the fractionated leaf lard/camellia oil fatty acids), 6% (w/w) of enzyme loading, and 6 h of reaction time at 45 °C. On the basis of gas chromatography determination and “deducting score” principle, a model was properly established for characterizing the quality of triacylglycerols enriched with 1,3-dioleoyl-2-palmitoylglycerol. This approach would be a valuable contribution in structured lipids industries because only gas chromatography determination was involved.

Fatty acid composition and distribution of human milk fat (HMF), from mothers over different lactating periods in Guangzhou, China, were analyzed. The universal characteristics were consistent with previously reported results although the fatty acid content was within a different range and dependent on the local population (low saturated fatty acid and high oleic acid for Guangdong mothers’ milk fat). Based on the composition of the total and sn-2 fatty acids of mature milk fat, an efficient evaluation model was innovatively established by adopting the “deducting score” principle. The model showed good agreement between the scores and the degree of similarity by assessing 15 samples from different sources including four samples of HMF, eight samples of human milk fat substitutes (HMFSs) and infant formulas, and three samples of fats and oils. This study would allow for the devolvement of individual human milk fat substitutes with different and specific fatty acid compositions for local infants.

Three commercial immobilized lipases, Lipozyme RM IM, Lipozyme TL IM and Novozym 435, were screened for the production of monoacylglycerols (MAG) by glycerolysis of camellia oil in a solvent medium of tert-butyl alcohol. Novozym 435 showed the best performance and was selected to catalyze the glycerolysis reaction. Different reaction conditions for the batch reaction, substrate mole ratio, substrate concentration and temperature, were investigated. The optimal reaction conditions were determined as 6:1 mole ratio of glycerol to camellia oil at 40% (w/v) of substrate concentration in tert-butyl alcohol at a reaction temperature of 50 °C. Under these optimal conditions, the conversion rate of camellia oil was 98.7% (10 h), and the mixture of acylglycerols contained 82.0% of MAG. A packed-bed reactor (PBR) system with 4.5 g Novozym 435 was employed in continuous production. The resulting product mixture of acylglycerols contained 80.74% of MAG and was obtained at a flow rate of 0.25 mL/min of substrates. The long-term operation of the PBR system gave an average productivity of 0.698 kg MAG/(kg enzyme h) after 38 days of operation.

Human milk fat substitute (HMFS) was prepared by Lipozyme RM IM-catalyzed acidolysis of lard and fatty acids obtained from palm kernel oil, tea seed oil and soybean oil, in a solvent-free system. The effects of substrate mass ratio, Lipozyme RM IM load and reaction time on the total fatty acids composition and sn-2 fatty acids composition were investigated. To optimize the reaction conditions, an orthogonal design was selected with three levels and three factors. Substrate mass ratio, Lipozyme RM IM load and reaction time were the factors employed. Under the given reaction temperature of 60 °C, the optimal reaction conditions were, 1/2 mass ratio of lard/fatty acids blend, 7% Lipozyme RM IM load and 1-h reaction time. By the “deducting score” principle, the produced HMFS in comparison with the reported HMFS possessed highest degree of similarity with local HMF from Guangzhou mothers. The results showed that it was possible to produce HMFS through the scale-up enzymatic acidolysis.

Exploration and evaluation of lipases from different sources will always be required by the swiftly developing industries for modification of oils and fats. Lipase SMG1 from Malassezia globosa was expressed in recombinant Pichia pastoris, and its catalytic activity in the hydrolysis and synthesis of partial glycerides was evaluated. Lipase SMG1 could not hydrolyze soybean oil; however, enhanced the hydrolysis of soybean oil by combining with Palatase 20000L, suggesting it showed strict specificity on mono- and diacylglycerol. Hydrolysates of soybean oil were esterified with glycerol to produce diacylglycerol, and the optimized results for the esterification reaction were with an fatty acids/glycerol molar ratio of 1:4, lipase SMG1 at a concentration of 120 U/g (U/w, with respect to total reactants), initial water content 1% (w/w, with respect to total reactants) and 30 °C, which yielded 62.03% of diacylglycerol in reaction mixture (80.5% in acylglyceride) after 12 h of reaction. The results showed that lipase SMG1 is a prospective enzyme which could be used in the oils and fats industry.Graphical abstractDownload full-size imageHighlights► Lipase from Malassezia globosa (lipase SMG1) was constitutively expressed in recombinant pichia pastoris. ► Lipase SMG1 could not hydrolyze soybean oil; however, enhanced the hydrolysis of soybean oil by combining with Palatase 20000L. ► Lipase SMG1 could effectively catalyze the esterification of soybean oil hydrolysates and glycerol to produce diacylglycerol (DAG), and 80.5% DAG in acylglycerol was obtained.

This paper presents a research interest concentrating on aims to establish a feasible industrial process for enzymatic production of highly pure glycerol monooleate (GMO). The synthesis of high oleic glycerol monooleate by enzymatic glycerolysis of high oleic sunflower oil, using Novozyme 435 as the biocatalyst, in a binary solvent mixture of tert-butanol and tert-pentanol (80/20, v/v), at a lab scale has been studied. A yield of 75.31% monoacylglycerol has been achieved at the first stage. A yield of 93.3% GMO was finally reached after further purification at the second stage. To evaluate the possibility of the process for industrialization, production of GMO was performed at a pilot-plant scale under the correspondingly adjusted conditions. A yield of 68.17% and 93.4% of GMO was obtained, respectively, at the end of the three stages.

Attempt was done to prepare food supplements with high content of c9, t11-CLA or t10, c12-CLA. A free acid mixture containing CLA isomers was esterified with ethanol by enzyme catalysis. Novozyme 435 and Lipase AY30 were screened, and Lipase AY30 was employed to catalyze esterification reaction because of its high fractionation efficiency. Effect of reaction conditions on total esterification was investigated, and the optimal reaction conditions were: 140 U of lipase amount, reaction temperature at 50 °C, a pH of 6.5, and molar ratio of FFA–CLA to ethanol at 1:1. Based on the studies above, experiments of esterification and purification were done, and the best fractionation efficiency was obtained when the total esterification was 37%, and the corresponding purity and recovery of c9, t11-CLA were 75.50 and 49.85%, and that of t10, c12-CLA were 72.02 and 62.32%.

•The substrate selectivity of AOL differed from most of lipases.•Structural model was constructed and elucidated the selectivity of AOL.•Conserved sequence motif F-X-X-H was common to RML-MDGL.•“Bridge-like” structure was a characteristic feature of RML-MDGL.Lipase from Aspergillus oryzae (AOL) was experimentally identified to be strictly specific for mono- and diacylglycerol, but not triacylglycerol, whose substrate selectivity was considerably different from most of lipases. The mechanism of this unique feature was still not clear. To address this problem, the structure of AOL in complex with substrate analog was constructed and molecular dynamics simulations were performed to fully relax steric clashes occurring in this enzyme-substrate complex. The bound diacylglycerol analog unambiguously identified two binding pockets, into which the corresponding parts of substrate were closely fitted. Comparison of AOL with the triacylglycerol lipase in homology revealed that its substrate selectivity mainly came from its unique binding pocket: a “bridge-like” structure was created by two bulky residues (W89 and F257) on the top of catalytic site, which prevented substrates with large size from entering the active site. In addition, a common feature that was responsible for substrate selectivity of mono- and diacylglycerol lipases (MDGL) was drawn. A well-conserved sequence motif F-X-X-H was identified, and this consensus sequence participated in substrate recognition of these MDGLs by forming the “bridge-like” structure that governed the specific binding of substrate. These results could provide a guide for discovering novel MDGLs and pave the way for site-directed mutagenesis experiments to improve enzyme for applications.Download full-size image

Research work was objectively targeted to synthesize highly pure diacylglycerol (DAG) with glycerolysis of soybean oil in a solvent medium of t-butanol. Three commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM and Novozym 435) were screened, and Novozym 435 was the best out of three candidates. Batch reaction conditions of the enzymatic glycerolysis, the substrate mass ratio, the reaction temperature and the substrate concentration, were studied. The optimal reaction conditions were achieved as 6.23:1 mass ratio of soybean oil to glycerol, 40% (w/v) of substrate concentration in t-butanol and reaction temperature of 50 °C. A two-stage molecular distillation was employed for purification of DAG from reaction products. Scale-up was attempted based on the optimized reaction conditions, 98.7% (24 h) for the conversion rate of soybean oil, 48.5% of DAG in the glycerolysis products and 96.1% for the content of DAG in the final products were taken in account as the results.

Highly efficient interfacial enzymatic hydrolysis of oil was achieved in a three-liquid-phase system, wherein the substrate constituted one of the phases. The enlarged interfacial area and relieved product inhibition were responsible for the high catalytic efficiency. Convenient product isolation and the high reusability of the enzyme were also demonstrated.