棕榈油脂肪酸蔗糖酯的合成研究

本文先采用棕榈油与甲醇反应制备脂肪酸甲酯,然后将所得脂肪酸甲酯与蔗糖在1,2-丙二醇体系中反应制备蔗糖酯。采用单因素法优化了蔗糖酯制备的反应条件,优化条件为：碳酸钾用量为反应物质量的23.5%、丙二醇用量为蔗糖质量的3.7倍、反应温度70℃和时间9h,在该优化条件下,蔗糖酯产率达70.8%。     

Production of L-erythrose via L-erythrulose from erythritol using microbial and enzymatic reactions

A rare aldotetrose, L-erythrose, was produced from erythritol via a two-step reaction. In the first step, complete oxidation of erythritol to L-erythrulose was achieved by using Gluconobacter frateurii IFO 3254. Washed cell suspension of the strain grown on tryptic soy broth (TSB) supplemented with 1% d-sorbitol was used to carry out the transformation reaction at 30 degrees C with shaking at 170 rpm. At 10% substrate concentration, 98% erythritol was converted to L-erythrulose within 48 h. The produced L-erythrulose was then used as a substrate for the production of L-erythrose. The isomerization of L-erythrulose to L-erythrose was carried out using constitutively produced L-ribose isomerase (l-RI) from the mutant strain Acinetobacter sp. DL-28 grown on D-lyxose mineral salt medium. At equilibrium, the yield of L-erythrose from L-erythrulose was 18% and finally 1.7 g L-erythrose was obtained from 10 g erythritol. After a number of simple purification steps, the product was isolated from the reaction mixture by ion-exchange column chromatography (Dowex 50W-X2, Ca2+). The structure of the product was determined after NaBH4 reduction from Infrared (IR) and 13C nuclear magnetic resonance (NMR) spectra.     

Alpha-anomer-selective glucosylation of (+)-catechin by the crude enzyme, showing glucosyl transfer activity, of Xanthomonas campestris WU-9701

Alpha-anomer-selective glucosylation of (+)-catechin was carried out using the crude enzyme, showing alpha-glucose transferring activity, of Xanthomonas campestris WU-9701 with maltose as a glucosyl donor. When 60 mg of (+)-catechin and 50 mg of the enzyme (5.25 units as maltose hydrolysing activity) were incubated in 10 ml of 10 mM citrate-Na2HPO4 buffer (pH 6.5) containing 1.2 M maltose at 45 degrees C, only one (+)-catechin glucoside was selectively obtained as a product. The (+)-catechin glucoside was identified as (+)-catechin 3'-O-alpha-D-glucopyranoside (alpha-C-G) by 13C-NMR, (1)H-NMR and two-dimensional HMBC analysis. The reaction at 45 degrees C for 36 h under the optimum conditions gave 12 mM alpha-C-G, 5.4 mg/ml in the reaction mixture, and the maximum molar conversion yield based on the amount of (+)-catechin supplied reached 57.1%. At 20 degrees C, the solubility in pure water of alpha-C-G, of 450 mg/ml, was approximately 100 fold higher than that of (+)-catechin, of 4.6 mg/ml. Since alpha-C-G has no bitter taste and a slight sweet taste compared with (+)-catechin which has a very bitter taste, alpha-C-G may be a desirable additive for foods, particularly sweet foods.     

Efficient production of L-(+)-lactic acid from raw starch by Streptococcus bovis 148

Streptococcus bovis 148 was found to produce L-(+)-lactic acid directly from soluble and raw starch substrates at pH 6.0. Productivity was highest at 37 degrees C, with 14.7 g/l lactic acid produced from 20 g/l raw starch. The yield and optical purity of L-lactic acid were 0.88 and 95.6%, respectively.     

Selective alpha-glucosylation of eugenol by alpha-glucosyl transfer enzyme of Xanthomonas campestris WU-9701

For one-step enzymatic synthesis of eugenyl alpha-glucoside as a promising pro-drug for a hair restorer and a derivative of spices, selective alpha-glucosylation of eugenol was carried out using the alpha-glucosyl transfer enzyme of Xanthomonas campestris WU-9701. When 130 micromol eugenol and crude enzyme showing 1.0 unit of alpha-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 8.0) containing 1.2 M maltose as a glucosyl donor at 40 degrees C, only one form of eugenyl glucoside was selectively obtained as a product and identified as eugenyl alpha-D-glucopyranoside (alpha-EG) by 13C-NMR, 1H-NMR, and two-dimensional heteronuclear multiple-bond coherence analyses. In the reaction, no other glucosylated products such as maltotriose or eugenyl maltoside were detected in the reaction mixture. The reaction at 40 degrees C for 48 h under the above conditions yielded 68 mumol alpha-EG in 2 ml suspension, and the maximum molar conversion yield based on the amount of eugenol supplied reached 52%.     

Human N-acetylglucosaminyltransferase I. Expression in Escherichia coli as a soluble enzyme, and application as an immobilized enzyme for the chemoenzymatic synthesis of N-linked oligosaccharides

N-Acetylglucosaminyltransferase I (GnT-I), which catalyzes the transfer of an N-acetylglucosamine residue from UDP-N-acetylglucosamine to the alpha1,3-linked mannose on Man5GlcNAc2 (M5), is a critical enzyme for the synthesis of high-mannose-type to complex-type glycan structures in N-linked glycan processing. We developed a large-scale preparation system for recombinant human GnT-I (hGnT-I) using the maltose binding protein (MBP) fusion system to facilitate the chemoenzymatic route for complex-type N-linked glycan synthesis. MBP-fused GnT-I was purified by affinity chromatography on an amylose resin column. The relative activity of MBP-fused GnT-I toward high-mannose-type N-linked oligosaccharides was 100% for Man5GlcNAc2, 52% for Man3GlcNAc2, 17% for Man6GlcNAc2. MBP-fused GnT-I exhibited optimal activity at pH 6.5-9.5 and was more active between pH 6.5-9.0. The optimum temperature for MBP-fused GnT-I activity was 40 degrees C, but the enzyme was active between 0-70 degrees C. Mn2+ and Co2+ were critical for the enzyme activity, while Zn2+ and Ca2+ inhibited the activity. Kinetic analysis of the purified enzyme showed an apparent K(m) value of 0.483 mM and a V(max) of 101 nmol/mg/min for M5. Immobilization of MBP-fused GnT-I on the amylose resin led to an 80% yield of the high mannose-type-of oligosaccharide.     

A novel enzymatic approach to the massproduction of L-galactose from L-sorbose

Wild-type strain of Pseudomonas cichorii ST-24 was unable to grow on D -psicose and inductively produced D -tagatose 3-epimerase (D -TE) with D -tagatose as an inducer. We have isolated a constitutive mutant, designated strain Ka75, which had acquired a new ability to grow on a mineral salts medium containing D -psicose as a sole carbon source. The D -psicose-metabolizing mutant synthesized a high level of D -TE. When grown on the culture medium supplemented with Mn(2+), the mutant strain produced around 250-fold higher activity than did the parent strain. Enzymatic properties of the constitutive enzyme were similar to those of the wild-type. Using the immobilized D -TE and recombinant L-rhamnose isomerase (L-RhI) from Escherichia coli strain JM109, a two-step enzymatic reaction was performed for massproduction of a rare aldo-hexose monosaccharide, L-galactose, from a common one, L-sorbose. In the first step, L-sorbose was epimerized to L-tagatose in a yield of 28%. The L-tagatose obtained was utilized as a starting material for L-galactose preparation by the immobilized L-RhI. At equilibrium, approximately 30% L-tagatose was isomerized to L-galactose. Finally, 7.5 g of L-galactose was obtained from 100 g of L-sorbose, viz an overall yield of 7.5%. The product obtained was purified and identified to be L-galactose by specific optical rotation and high performance liquid chromatography (HPLC) analysis, and was ultimately confirmed by (13)C nuclear magnetic resonance ((13)C NMR) and IR spectra.     

α-Anomer-selective glucosylation of (+)-catechin by the crude enzyme, showing glucosyl transfer activity, of Xanthomonas campestris WU-9701

α-Anomer-selective glucosylation of (+)-catechin was carried out using the crude enzyme, showing α-glucose transferring activity, of Xanthomonas campestris WU-9701 with maltose as a glucosyl donor. When 60 mg of (+)-catechin and 50 mg of the enzyme (5.25 units as maltose hydrolysing activity) were incubated in 10 ml of 10 mM citrate-Na₂HPO₄ buffer (pH 6.5) containing 1.2 M maltose at 45°C, only one (+)-catechin glucoside was selectively obtained as a product. The (+)-catechin glucoside was identified as (+)-catechin 3′-O-α- -glucopyranoside (α-C-G) by ¹³C-NMR, ¹H-NMR and two-dimensional HMBC analysis. The reaction at 45°C for 36 h under the optimum conditions gave 12 mM α-C-G, 5.4 mg/ml in the reaction mixture, and the maximum molar conversion yield based on the amount of (+)-catechin supplied reached 57.1%. At 20°C, the solubility in pure water of α-C-G, of 450 mg/ml, was approximately 100 fold higher than that of (+)-catechin, of 4.6 mg/ml. Since α-C-G has no bitter taste and a slight sweet taste compared with (+)-catechin which has a very bitter taste, α-C-G may be a desirable additive for foods, particularly sweet foods.     

Enzymatic synthesis of alpha-arbutin by alpha-anomer-selective glucosylation of hydroquinone using lyophilized cells of Xanthomonas campestris WU-9701

Alpha-arbutin, a useful cosmetic ingredient, was selectively synthesized by alpha-anomer-selective glucosylation of hydroquinone with maltose as a glucosyl donor using lyophilized cells of Xanthomonas campestris WU-9701 as a biocatalyst. When 45 mM hydroquinone and 120 mg of lyophilized cells showing 11 nkat of alpha-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3NaOHKCl buffer (pH 7.5) containing 1.2 M maltose at 40 degrees C, only one form of hydroquinone glucoside was selectively obtained as a product and identified as hydroquinone 1-O-alpha-D-glucopyranoside (alpha-arbutin) by 13C-NMR, 1H-NMR and two-dimensional HMBC analysis. Although hydroquinone has two phenolic -OH groups at the para position in its structure, only one -OH group, but not both -OHs, was glucosylated and no other glucosylated products such as maltotriose were detected in the reaction mixture. The reaction at 40 degrees C for 36 h under optimum conditions yielded 42 mM alpha-arbutin, and the maximum molar conversion yield based on the amount of hydroquinone supplied reached 93%.     

Supercritical Carbon Dioxide Extraction of Valuable Compounds from Citrus junos Seed

Extraction of Citrus junos seed was carried out at temperatures of 40–70 °C, pressures of 20–50 MPa, and CO₂ flow rate of 3 ml/min with supercritical carbon dioxide to obtain the valuable compounds. Seed oil was also extracted by using Soxhlet extraction with hexane as the solvent during 360 min for comparison with the efficiency of supercritical carbon dioxide extraction. Gas chromatography–mass spectrometry (GC–MS) was used to analyze the components present in the seed oil and Gas chromatography-flame ionization detector (GC-FID) was used to quantify their amounts. Among the conditions studied, the highest extraction yield was obtained at higher pressure and temperature (50 MPa and 70 °C). The extraction yield was about 29.5% of the seed, which was almost comparable to that of hexane Soxhlet extraction (33.8%). The results of the GC–MS analyses showed that the seed oil extracted contained N-methylanthranyl acid methyl, fatty acids (such as palmitic, stearic, oleic, linoleic, and linolenic acid), and physiologically active substances of β-sitosterol and squalene.     

Production of high degree polymerized chitooligosaccharides in a membrane reactor using purified chitosanase from Bacillus cereus

Crude chitosanase from Bacillus cereus NTU-FC-4 was separated by a cation exchanger to three fractions named CBCI, CBCII, and CBCIII. The CBCI hydrolyzed chitosan to yield dimers. The primary hydrolytic products of CBCII were low degree polymerized (DP) chitooligosaccharides. The CBCIII had the fastest reaction rate and yielded high DP chitooligosaccharides (heptamer and higher DP oligomers). When CBCIII was used in the ultrafiltration membrane reactor with enzyme/substrate ratio 0.06 unit/mg and 100 min of residence time (RT), the concentration of high DP oligomers was 9.78 mg/mL which occupied ca. 48% of total oligomers in the final product as compared to ca. 29% resulted from the crude enzyme. Decrease of RT to 50 min and 33 min, the high DP oligomers in the products were ca. 61% and 69%, respectively. This system could be operated for at least 24 h and kept a constant permeate flux and product output rate.     

Selection of entomopathogenic fungi for aphid control

Twelve strains of entomopathogenic fungi such as Lecanicillium lecanii, Paecilomyces farinosus, Beauveria bassiana, Metarhizium anisopliae, Cordyceps scarabaeicola, and Nomuraea rileyi were screened for aphid control. At 25 degrees C and 75% relative humidity (RH), among tested entomopathogenic fungi, L. lecanii 41185 showed the highest virulent pathogenicity for both Myzus persicae and Aphis gossypii, and their control values were both nearly 100% 5 and 2 d after treatment, respectively. Moreover, at an RH of 45% and in a wide temperature range (20-30 degrees C), L. lecanii 41185 also exhibited the highest virulence to M. persicae. The control value of M. persicae and the 50% lethal time (LT50) decreased significantly as the applied conidial concentration increased. The 50% lethal concentration (LC50) of the conidial suspension of this fungus was determined to be 6.55x10(5) conidia/ml. The control values of M. persicae resulting from the application of 1x10(7) and 1x10(8) conidia/ml were nearly the same and were significantly higher than that of 1x10(6) conidia/ml. The tested entomopathogenic fungi grew in a broad temperature range (15-30 degrees C). Lecanicillium strains showed optimum growth at 25 degrees C. The aerial conidia of Lecanicillium strains also could germinate in a broad temperature range (15-30 degrees C) and L. lecanii 41185 was the only strain with conidial germination at 35 degrees C.     

A new microbial method for more efficient production of Nalpha-benzyloxycarbonyl-L-aminoadipate delta-semialdehyde and Nalpha-benzyloxycarbonyl-D-aminoadipate delta-semialdehyde

A new biochemical method for more efficient production of Nalpha-benzyloxycarbonyl-L-aminoadipate delta-semialdehyde (Nalpha-Z-L-AASA) and Nalpha-Z-D-AASA was developed with cells of Rhodococcus sp. AIU Z-35-1. Using the cells harvested after 1 d of cultivation, more than 95 mM Nalpha-Z-L-AASA was produced from 100 mM Nalpha-Z-L-lysine by incubating at pH 5.0 for 1 d at 30 degrees C or by incubating at pH 7.0 for 2 d at 10 degrees C. A similar conversion yield of Nalpha-Z-D-AASA was also obtained under the same conditions. These reaction times required were 1/4 and 1/2 of the respective ones by the method with amine oxidase, and the yields of Nalpha-Z-L-AASA and Nalpha-Z-D-AASA were 2 times higher than the respective ones by the method with amine oxidase. In addition, this method had the advantages of not requiring purification of enzyme and addition of catalase. Thus, the microbial method proposed here was superior to the chemical and other biochemical methods in simplicity, reaction rate, and yield.     

Solvent-free production of 1,3-diglyceride of CLA: Strategy consideration and protocol design

Enzymatic production of a homogeneous 1,3-diglyceride of polyunsaturated fatty acids (PUFAs) was carried out using Novozym 435 as biocatalyst and conjugated linoleic acid (CLA) as a model fatty acid. Three different operation modes, namely, magnetic stirring under vacuum, vacuum-driven N₂ bubbling and incubation with molecular sieves, were examined to find an efficient protocol for the enzymatic production. Studies on the effects of mass transfer showed that the occurrence of mass transfer limitation was strongly dependent on the operational modes. Vacuum-driven N₂ bubbling proved to be capable of eliminating mass transfer resistance, creating effective interaction for a multiple-phase reaction system and yielding an efficient water removal and a faster reaction rate. Hence, vacuum-driven N₂ stirring was considered as the best choice among the tested strategies for the production of pure 1,3-diglyceride of PUFAs with industrial interests, because it gave a higher yield of the desired product, higher productivity and lower impurity content due to its suppression of the acylmigration of 1,3-diglyceride to 1,2-diglyceride. The yield of 92–96% 1,3-dCLG could be obtained when 5 mmol of glycerol were incubated with 10–12 mmol CLA for about 3 h at 45–55 °C and a pressure less than 10 mbar, with enzyme loading of 40–70 g l⁻¹. Among the operational parameters, temperature and reaction time were found to have profound effects on the acylmigration and yield of 1,3-diglyceride. Moreover, the enzyme showed excellent operational stability in this protocol under the optimized conditions (little activity loss of enzyme was observed after 10 consecutive batch reactions), indicating the potential of this technology for industrial application.     

Valorization of Olive Pomace Oil with Enzymatic Synthesis of 2‐Monoacylglycerol

2‐Monoacylglycerols (2‐MAG) with a high content of oleic acid at sn‐2 position was synthesized by enzymatic ethanolysis of refined olive pomace oil, which is a byproduct of olive oil processing. Six lipases from different microbial sources were used in the synthesis of 2‐MAG. Immobilized lipase from Candida antarctica gave the highest product yield among the selected lipases. Response surface methodology was applied to optimize reaction conditions; time (4 to 10 h), temperature (45 to 60 °C), enzyme load (10 to 18 wt%), and ethanol:oil molar ratio (30:1 to 60:1). The predicted highest 2‐MAG yield (84.83%) was obtained at 45 °C using 10 (wt%) enzyme load and 50:1 ethanol:oil molar ratio for 5 h reaction time. Experiments to confirm the predicted results at optimum conditions presented a 2‐MAG yield of 82.54%. The purification yield (g 2‐MAG extracted/100 g of total product) was 80.10 and 69.00 for solvent extraction and low‐temperature crystallization, respectively. The purity of the synthesized 2‐MAG was found to be higher than 96%.     

离子型反丁烯二酸单甲酯的合成及抑菌活性研究

以顺丁烯二酸酐和甲醇为原料合成了反丁烯二酸单甲酯,通过正交试验考察了催化剂的用量、异构化时间及异构化温度对产率的影响。结果表明,反应原料顺丁烯二酸酐与甲醇物质的量比为1.1:1(摩尔比),在60℃下反应1h 实现单酯化后,加入10.9% 的三氯化铝在80℃下进行转型2h,产率为71.9%。并通过交叉融合法合成了离子型反丁烯二酸单甲酯,用熔点测定法、红外光谱法和NMR 对产品进行了鉴定。同时采用比浊法、平板法测定离子型反丁烯二酸单甲酯对细菌、霉菌、酵母的抑菌效果。结果表明离子型反丁烯二酸单甲酯对它们均有较强的抑菌活性,有望开发成为新型抑菌剂。     

Acetate and ethanol production from H2 and CO2 by Moorella sp. using a repeated batch culture

The growth inhibition of Moorella sp. HUC22-1 by undissociated acetic acid was analyzed using a non-competitive inhibition model coupled with a pH inhibition model. In the cells grown on H2 and CO2, the inhibition constant, K(p) of the undissociated acetic acid was 6.2 mM (164 mM as the total acetate at pH 6.2, pKa = 4.795, 55 degrees C), which was 1.5-fold higher than that obtained in cells grown on fructose. When a pH-controlled batch culture was performed using a fermentor at pH 6.2 with H2 and CO2, a maximum of 0.92 g/l of dry cell weight and 339 mM of acetate were produced after 220 h, which were 4.4- and 6.8-fold higher than those produced in the pH-uncontrolled batch culture, respectively. In order to reduce acetate inhibition in the culture medium, a repeated batch culture with cell recycling was performed at a constant pH with H2 and CO2. At a pH of 6.2, the total acetate production reached 840 mmol/l-reactor with 4.7 mmol/l-reactor of total ethanol production after 420 h. When the culture pH was maintained at 5.8, which was the optimum for ethanol production, the total ethanol production reached 15.4 mmol/l-reactor after 430 h, although the total acetate production was decreased to 675 mmol/l-reactor.     

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27°C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.     

黑加仑果酒酿造工艺的研究

黑加仑果酒是以黑加仑为主要原料,经过液态发酵制得的一种新型饮品。通过对果胶酶水解、酒精发酵过程的研究,最终确定了黑加仑果酒的酿造工艺。结果表明,使用0.55%果胶酶在35℃水解3h,黑加仑的出汁率为89.53%;黑加仑汁中添加50mg/L的SO2,然后接入活化4h的活性干酵母,30℃发酵7d,酒精度可达7.94%vol,酿制出的黑加仑果酒果香突出、口味柔和。     

Transformation of cellobiose to 3-ketocellobiose by the EDTA-treated Agrobacterium tumefaciens cells

Agrobacterium tumefaciens cells treated with EDTA were used as a biocatalyst for the transformation of cellobiose to 3-ketocellobiose. The effects of pH of the reaction medium and temperature on the reaction rate and the selectivity during the transformation by the cells were examined, and it was found that the optimum reaction conditions were pH 6 and 15 degrees C. The selectivity was about 0.8 under these conditions. The substrate concentration had no significant effect on the selectivity up to 300 mmol/I.