Enhancing immobilization of Aspergillus oryzae ST11 lipase on polyacrylonitrile nanofibrous membrane by bovine serum albumin and its application for biodiesel production.

The partially purified lipase from Aspergillus oryzae ST11 was immobilized on the surface-modified electrospun polyacrylonitrile (PAN) nanofibrous membrane. The effects of time and concentrations of glutaraldehyde and bovine serum albumin (BSA) were studied. The immobilized lipase reached the maximum activity at 180 min with the recovered activity of 44.2% (0.22 U/mg-support) in the absence of BSA. After adding 6 mg/mL BSA and 40 mM glutaraldehyde gave the recovered activity at 86.9% (0.43 U/mg-support). It showed the highest stability at pH 7.5 but the soluble lipase was at pH 7.0. In addition, the immobilized lipase was more stable at pH 4.0 and 9.0. The immobilized lipase preserved 56% activity at 70 °C but the soluble lipase had 34% activity. It conserved 86% activity while the soluble lipase had 18% activity after 13 days of storage at 4 °C. The operating parameters such as biocatalyst concentration, water content, and the molar ratio of methanol and palm oil were optimized for biodiesel production. The highest biodiesel conversion (95%) was obtained with the immobilized lipase 15% (w/w), 40% (w/w) 50 mM Tris-HCl buffer pH 7.0 and methanol/palm oil (3.5:1.0). This immobilized lipase could be reused with 82.9% conversion after 10 cycles of batch production.

Enzymatic synthesis of coconut oil based wax esters by immobilized lipase EQ3 and commercial lipozyme RMIM

BACKGROUND: Liquid wax esters are widely used in cosmetic as well as pharmaceutical and other industries. The demand of organic and natural products is increasing nowadays. Coconut oil contains benefit fatty acids and has been mainly used for oil-based and moisturizer products. Liquid wax esters from coconut oil and unsaturated fatty alcohol can be synthesized by enzymatic reaction; and it is interesting for using as an alternative natural ingredient in these industries. RESULTS: Optimal condition for coconut oil based wax ester synthesis by immobilized lipase EQ3 was 10 U of enzyme, temperature at 30°C and molar ratio of coconut oil to oleyl alcohol at 1:3 (mol/mol) (0.33X) dissolved in isooctane for 12 h, while for Lipozyme RM IM optimal condition was 10 U of enzyme, temperature at 45°C and oil/alcohol molar ratio at 1:3 (0.33X) dissolved in isooctane for 3 h. Percentage of wax esters synthesized by both lipases reached more than 88%. Both immobilized lipases catalyzed high yield of wax esters within the 2nd batch; after that, the immobilized lipases showed reduced activity and synthesized b60% of wax esters from the 3rd to 5th batch. The main composition of wax esters was ~48% oleyl laurate with 10% degradation at ~250°C. CONCLUSIONS: The liquid wax ester synthesis by commercial Lipozyme RM IM had higher effect than immobilized lipase EQ3, but both catalysts were stable within 2 batches in the optimum condition. The characteristic properties of wax esters showed potential for use as components in cosmetics and skin care products.

Magnetic Cross-Linked Enzyme Aggregates of Aspergillus oryzae ST11 Lipase Using Polyacrylonitrile Coated Magnetic Nanoparticles for Biodiesel Production.

The biodiesel production by enzymatic catalysis is the environmentally friendly production process. In this work, the polyacrylonitrile coated magnetic nanoparticles were prepared and used as a core supporter for producing magnetic cross-linked enzyme aggregates (mCLEAs) from Aspergillus oryzae ST11 lipase with the co-feeding of bovine serum albumin (BSA). The highest immobilized lipase activity was 0.09 U/mg-support or 81.7% of recovered activity. The resulting mCLEAs exhibited the desired characteristics; it had improved the stabilities for pH, temperature, as well as storage comparing to free enzyme. Moreover, when this biocatalyst was used for biodiesel production at the optimal conditions (30% w/w of mCLEAs, 30% w/w of water content and stepwise addition of methanol), the biodiesel conversion was 95% within 24 h of reaction, while one-step addition of methanol produced 81% conversion. The mCLEAs could be reused for 5 cycles and retained the biodiesel conversion higher than 60%.

Optimization of Cellulase and Xylanase Productions by Streptomyces thermocoprophilus Strain TC13W Using Oil Palm Empty Fruit Bunch and Tuna Condensate as Substrates.

The modified medium composed of the alkaline-pretreated oil palm empty fruit bunch (APEFB) and tuna condensate powder was used for cellulase and xylanase productions by Streptomyces thermocoprophilus strain TC13W. The APEFB contained 74.46% (w/w) cellulose, 15.72% (w/w) hemicellulose, and 6.40% (w/w) lignin. The tuna condensate powder contained 55.49% (w/w) protein and 11.05% (w/w) salt. In the modified medium with only 6.75 g/l tuna condensate powder, 10 g/l APEFB, and 0.5 g/l Tween 80, S. thermocoprophilus strain TC13W produced cellulase 4.9 U/ml and xylanase 9.0 U/ml. The enzyme productions in the modified medium were lower than cellulase (6.0 U/ml) and xylanase (12.0 U/ml) productions in the complex medium (CaCl2 0.1, MgSO4·7H2O 0.1, KH2PO4 0.5, K2HPO4 1.0, NaCl 0.2, yeast extract 5.0, NH4NO3 1.0, Tween 80 0.5). When tuna condensate powder in the modified medium was reduced to 5.0 g/l and Tween 80 was increased to 1.5 g/l, S. thermocoprophilus strain TC13W produced cellulase and xylanase activities of 9.1 and 12.1 U/ml, respectively. This study shows that the cost of enzyme production could be reduced by using pretreated EFB and tuna condensate as a carbon and a nitrogen source, respectively.

Characterization of an α-amino-ɛ-caprolactam racemase with broad substrate specificity from Citreicella sp. SE45.

α-Amino-ε-caprolactam (ACL) racemizing activity was detected in a putative dialkylglycine decarboxylase (EC 4.1.1.64) from Citreicella sp. SE45. The encoding gene of the enzyme was cloned and transformed in Escherichia coli BL21 (DE3). The molecular mass of the enzyme was shown to be 47.4 kDa on SDS-polyacrylamide gel electrophoresis. The enzymatic properties including pH and thermal optimum and stabilities were determined. This enzyme acted on a broad range of amino acid amides, particularly unbranched amino acid amides including L-alanine amide and L-serine amide with a specific activity of 17.5 and 21.6 U/mg, respectively. The K m and V max values for D- and L-ACL were 5.3 and 2.17 mM, and 769 and 558 µmol/min.mg protein, respectively. Moreover, the turn over number (K cat) and catalytic efficiency (K cat/K m ) of purified ACL racemase from Citreicella sp. SE45 using L-ACL as a substrate were 465 S-1 and 214 S-1mM-1, respectively. The new ACL racemase from Citreicella sp. SE45 has a potential to be used as the biocatalytic application.

Water footprints of products of oil palm plantations and palm oil mills in Thailand.

The water footprint (WF) of fresh fruit bunches (FFBs) from oil palm plantations and crude palm oil (CPO) from palm oil mills in southern and eastern Thailand were determined over 25 years. Climatic conditions, soil characteristics, and the characteristics of oil palm growth were considered. The WF of FFBs was 1063 m(3)/ton (t) on average. Green, blue, and grey waters comprised of 68, 18, and 14% of total WF, respectively. The oil palm plantations in Thailand required smaller amounts of indirect blue water. The average WF for producing a ton of CPO of seven mills was 5083 m(3). Most of the waters used in the mills originated from indirect green, blue and grey waters from the plantations. The direct blue water used in the mills had less impact on the total WF, lower than 1% of the total WF. Average percentages of green, blue, and grey waters of 69, 16, and 15% of total WF were determined for the mills, respectively. The water deprivation of the FFBs and CPO ranged from 0.73-12.9 and 3.44-58.3 m(3)H2Oeq/t, respectively. In 2013, the CPO production in Thailand including green, blue, and grey waters from plantation and blue water from mills required 11,343 million m(3) water. If the oil palm variety Suratthani 7 is used in the plantation, it would increase the yield from 15.2 to 22.8 t FFBs/ha-year and decrease the WF to 888 m(3)/t FFBs. The average value of the oil extraction rate (OER) of mills was 18.1%. With an increase in the OER of 1%, a reduction of the WF of 250 m(3)/t CPO or 5.1% of total WF could be obtained.

In Silico Identification for α-Amino-ε-Caprolactam Racemases by Using Information on the Structure and Function Relationship.

In silico identification for enzymes having desired functions is attractive because there is a possibility that numerous desirable enzymes have been deposited in databases. In this study, α-amino-ε-caprolactam (ACL) racemases were searched from the NCBI protein database. Four hundred thirteen fold-type I pyridoxal 5'-phosphate-dependent enzymes which are considered to contain sequences of ACL racemase were firstly obtained by submitting the sequence of ACL racemase from Achromobacter obae to the database. By identifying Lys241 as a key amino acid residue, 13 candidates for ACL racemase were selected. Then, putative ACL racemase genes were synthesized as codon-optimized sequences for expression in Escherichia coli. They were subcloned and expressed in E. coli BL21 and underwent His-tag purification. ACL and amino acid amide racemizing activities were detected among ten of the candidates. The locus tags Oant_4493, Smed_5339, and CSE45_2055 derived from Ochrobactrum anthropi ATCC49188, Sinorhizobium medicae WSM 419, and Citreicella sp. SE45, respectively, showed higher racemization activity against D- and L-ACLs rather than that of ACL racemase from A. obae. Our results demonstrate that the newly discovered ACL racemases were unique from ACL racemase from A. obae and might be useful for applications in dynamic kinetic resolution for D- or L-amino acid production.

Incorporation of nisin Z and lauric arginate into pullulan films to inhibit foodborne pathogens associated with fresh and ready-to-eat muscle foods.

A combination of food grade compounds with edible films, used to inhibit foodborne pathogens associated with fresh or further processed muscle foods, is receiving considerable attention. In this study, pullulan films containing lauric arginate (LAE) and nisin Z (produced by Lactococcus lactis subsp. lactis I8-7-3 and isolated from catfish gut), alone or in combination, were investigated for controlling foodborne pathogens on fresh and further processed muscle foods after long-term refrigerated storage. Salmonella Typhimurium and Salmonella Enteritidis on raw turkey breast slices wrapped with a film containing LAE or the combination of LAE with nisin Z were reduced throughout the experiment, 2.5 to 4.5 log10 CFU/cm(2) and 3.5 to 5.1 log10 CFU/cm(2), respectively. Film containing a combination of LAE with nisin Z reduced Staphylococcus aureus and Listeria monocytogenes Scott A inoculated onto ham surfaces by approximately 5.53 and 5.62 log10 CFU/cm(2), respectively during refrigerated storage. Escherichia coli O157:H7, O111, and O26 also were reduced by >4 log 10CFU/cm(2) on raw beef slices after treatment with the combination film and refrigerated storage. The results obtained from this study indicate the LAE- and LAE-nisin Z-containing pullulan films displayed excellent inhibition against foodborne pathogens on fresh and further processed muscle foods.

Effect of lauric arginate, nisin Z, and a combination against several food-related bacteria.

The effects of lauric arginate (LAE) and nisin Z, alone or in combination, on cell damage were investigated against Escherichia coli O157:H7, Listeria monocytogenes and Brochothrix thermosphacta, by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations, efflux of potassium and phosphate ions, and growth inhibition. A combination of LAE with nisin Z caused severe and dramatic changes in the cytoplasmic membrane and cell lysis of both Gram-positive and Gram-negative bacteria. The combination treatment also caused significant potassium and phosphate ion leakage of E. coli O157:H7, L. monocytogenes and B. thermosphacta, when compared with other treatments: 16.62±1.05, 50.35±0.81 and 45.47±1.15mg/L of potassium ion and 122.66±8.81, 97.96±3.31 and 26.47±13.97mg/L of phosphate ion after treatment for 6h, respectively. Bacteria were reduced by approximately 7log10CFU/mL within the first hour of treatment and then cells were unable to grow for the remainder of the experiment. Treatment with LAE alone resulted in changes in cellular morphology, coagulation of the cytoplasm, and low level leakage of potassium and phosphate ions in all bacteria tested. Treatment of L. monocytogenes and B. thermosphacta with nisin Z (320AU/mL of final concentration) resulted in the formation of membrane channels and leakage of potassium and phosphate ions at rather high levels; but the bacteriocin was not effective against E. coli O157:H7. LAE or nisin Z reduced growth of both L. monocytogenes and B. thermosphacta by approximately 7log10CFU/mL. Conversely, E. coli O157:H7 was not inhibited by treatments with nisin Z, but decreased by approximately 4.45log10CFU/mL after treatment with LAE. These findings provide additional information on the mode of action of these compounds on bacterial populations.

Alternative technologies for the reduction of greenhouse gas emissions from palm oil mills in Thailand.

Alternative methodologies for the reduction of greenhouse gas (GHG) emissions from crude palm oil (CPO) production by a wet extraction mill in Thailand were developed. The production of 1 t of CPO from mills with biogas capture (four mills) and without biogas capture (two mills) in 2010 produced GHG emissions of 935 kg carbon dioxide equivalent (CO2eq), on average. Wastewater treatment plants with and without biogas capture produced GHG emissions of 64 and 47% of total GHG emission, respectively. The rest of the emissions mostly originated from the acquisition of fresh fruit bunches. The establishment of a biogas recovery system must be the first step in the reduction of GHG emissions. It could reduce GHG emissions by 373 kgCO2eq/t of CPO. The main source of GHG emission of 163 kgCO2eq/t of CPO from the mills with biogas capture was the open pond used for cooling of wastewater before it enters the biogas recovery system. The reduction of GHG emissions could be accomplished by (i) using a wastewater-dispersed unit for cooling, (ii) using a covered pond, (iii) enhancing the performance of the biogas recovery system, and (iv) changing the stabilization pond to an aerated lagoon. By using options i-iv, reductions of GHG emissions of 216, 208, 92.2, and 87.6 kgCO2eq/t of CPO, respectively, can be achieved.

Providencia thailandensis sp. nov., isolated from seafood processing wastewater.

The bacterial strain C1112(T) was isolated from seafood processing wastewater collected from a treatment pond of the seafood factory in Songkhla Province, Thailand. Phylogenetic analysis based on concatenated sequences from the 16S rRNA gene and five housekeeping genes, fusA, lepA, leuS, gyrB and ileS respectively showed that the strain C1112(T) belonged to the genus Providencia, and share 91.75% similarity with P. stuartii DSM 4539(T). DNA-DNA hybridization between the strain C1112(T) and P. stuartii KCTC 2568(T) was 48.1% relatedness. Moreover, some results from biochemical properties indicated that the strain C1112(T) was distinguished from the phylogenetically closest relatives. The major fatty acids of the strain C1112(T) were C16:0, iso-C15:0, C14:0 and C17:0 cyclo and the DNA G+C content was 41 mol%. Based on the genotypic and phenotypic considerations, it should be classified as a novel species of the genus Providencia for which the name Providencia thailandensis sp. nov. is proposed. The type strain is C1112(T) (= KCTC 23281(T) =NBRC 106720(T)).

Enterobacter siamensis sp. nov., a transglutaminase-producing bacterium isolated from seafood processing wastewater in Thailand.

A novel strain of Enterobacter, C2361(T), a Gram-negative, non-spore-forming, rod-shaped and facultative anaerobic bacterium with the capability to produce transglutaminase, was isolated from seafood processing wastewater collected from a treatment pond of a seafood factory in Songkhla Province, Thailand. Phylogenetic analyses and phenotypic characteristics, including chemotaxonomic characteristics, showed that the strain was a member of the genus Enterobacter. The 16S rRNA gene sequence similarities between strain C2361(T) and Enterobacter cloacae subsp. cloacae ATCC 13047(T) and Enterobacter cloacae subsp. dissolvens LMG 2683(T) were 97.5 and 97.5%, respectively. Strain C2361(T) showed a low DNA-DNA relatedness with the above-mentioned species. The major fatty acids were C16:0, C17:0cyclo and C14:0. The DNA G+C content was 53.0 mol%. On the basis of the polyphasic evidence gathered in this study, it should be classified as a novel species of the genus Enterobacter for which the name Enterobacter siamensis sp. nov. is proposed. The type strain is C2361(T) (= KCTC 23282(T) = NBRC 107138(T)).

Bacteriocin-Producing Lactic Acid Bacteria Isolated from Mangrove Forests in Southern Thailand as Potential Bio-Control Agents: Purification and Characterization of Bacteriocin Produced by Lactococcus lactis subsp. lactis KT2W2L.

The aim of this work was to purify and characterize the bacteriocin produced by Lactococcus lactis subsp. lactis KT2W2L previously isolated from mangrove forests in southern Thailand, in order to evaluate its potential as new food protective agent. The active peptide from the cell-free supernatant of this strain was purified in 4 steps: (1) precipitation with 70 % saturated ammonium sulfate, (2) elution on a reversed-phase cartridge using different concentrations of acetonitrile, (3) cation-exchange chromatography and (4) final purification by reversed-phase HPLC on a C8 column. The molecular mass of 3,329.5254 Da of the purified bacteriocin, determined by mass spectrometry, is nearly identical to that of peptide nisin Z. The activity of the purified bacteriocin was unaffected by pH (2.0-10.0), thermostable but was sensitive to proteolytic enzymes. The bacteriocin activity was stable after 8 weeks of storage at -20 °C and 7 weeks of storage at 4 °C, but decreased after 3 weeks of storage at 37 °C. It was stable when incubated for 1 month at 4 °C in 0-30 % NaCl. Inhibitory spectrum of this bacteriocin showed a wide range of activity against similar bacterial strains, food-spoilage and food-borne pathogens. L. lactis subsp. lactis KT2W2L was sensitive to kanamycin, penicillin and tetracycline but resistant to ampicillin, gentamicin and vancomycin. The fragment obtained after amplification of genomic DNA from L. lactis subsp. lactis KT2W2L, with specific primers for bacteriocin genes, presented 99 % homology to the nisin Z gene. PCR amplification demonstrated that L. lactis subsp. lactis KT2W2L does not harbor virulence genes cylA, cylB, efaAfs and esp. The bacteriocin and its producing strain may find application as bio-preservatives for reduction in food-spoilage and food-borne pathogens in food products.

Hydrolysis of surimi wastewater for production of transglutaminase by Enterobacter sp. C2361 and Providencia sp. C1112.

Surimi wastewater (SWW) is an industrial wastewater, released during the washing step of surimi preparation from minced fish, that causes environmental problem. In this study, SWW produced from ornate threadfin bream (Nemipterus hexodon) was hydrolysed and used to cultivate Enterobacter sp. C2361 and Providencia sp. C1112 for the production of microbial transglutaminase (MTGase, EC 2.3.2.13). The SWW was repeatedly used to wash the fish mince that gained a final protein content of 3.20% (w/v). The commercial protease, Delvolase was the most appropriate protease used to produce fish protein hydrolysate (FPH) from SWW. The FPH at 40% degree of hydrolysis was used instead of a peptone portion in the SPY medium (3.0% starch, 2.0% peptone, 0.2% yeast extract, 0.2% MgSO(4), 0.2% K(2)HPO(4) and 0.2% KH(2)HPO(4), pH 7.0) to cultivate the tested strains at 37°C, shaking speed at 150rpm. Providencia sp. C1112 produced higher MTGase activity (1.78±0.05U/ml) than Streptoverticillium mobaraense (1.61±0.02U/ml) at 18h of cultivation in FPH medium. On the other hand, the Enterobacter sp. C2361 produced lower MTGase activity (1.18±0.03U/ml).

Hydroxamate-based colorimetric method for direct screening of transglutaminase-producing bacteria.

Microbial transglutaminase (MTGase) is a commercial enzyme that has been applied to many protein containing foods to improve their textural property. The screening of MTGase-producing microorganisms from various sources might lead to the discovery of a new MTGase with different characteristics. This report demonstrates the use of a direct detection method for MTGase-producing bacteria grown on an agar plate by filter paper disc (FPD) assay. The principle of the assay is the formation of a red burgundy color by the hydroxamate-ferric complex. The color developed intensity was linearly correlated by the concentration of hydroxamic acid in the range of 0.1-0.8 µM and was visually scored at 4 levels: 0, 1, 2 and 3. Streptoverticillium mobaraense DSM 40847, a positive MTGase-producer, was chosen for the verification and improving of the proposed method. The colonies grown on the nutrient agar plate at 37°C for 24 h were covered with FPDs and 30 µl of substrates (CBZ-Gln-Gly and hydroxylamine). After incubation, 10 µl of the ferric-TCA-HCl solution was placed on the FPD. The optimal time taken to catalyze the formation of CBZ-Gln-Gly-hydroxamic acid by the MTGase and the time taken for the hydroxamate-ferric complex to form color were 180 and 60 min, respectively. Using this assay, 30 of 189 colonies isolated from wastewater and floating-floc samples showed MTGase-positive colonies which were well correlated to the quantitative screening of MTGase activity (R(2) = 0.9758). The results revealed that the FPD assay could be used for the qualitative screening of MTGase-producing bacteria.

Comparative study on protein cross-linking and gel enhancing effect of microbial transglutaminase on surimi from different fish.

BACKGROUND: Microbial transglutaminase (MTGase) has been used to increase the gel strength of surimi. Nevertheless, its effectiveness varies with fish species. The aim of this study was to elucidate the effect of MTGase at different levels on protein cross-linking and gel property of surimi from threadfin bream, Indian mackerel and sardine in the presence and absence of endogenous transglutaminase. RESULT: Breaking force of all surimi gels increased as MTGase levels (0-0.6 U g⁻¹) increased except for threadfin bream surimi gel, where the breaking force decreased at 0.6 U g⁻¹ (P < 0.05). In the presence of EDTA, the gel strengthening effect was lower, suggesting the combined effect of endogenous transglutaminase with MTGase. With the addition of MTGase, the gel with the highest increase in breaking force showed highest decrease in myosin heavy chain. When cross-linking activity of MTGase on natural actomyosin (NAM) was determined, the highest decreasing rate in ε-amino group content with the concomitant increased formation of cross-linked proteins was found in NAM from threadfin bream. The reactivity of muscle proteins toward MTGase-induced cross-linking was in agreement with surimi gel strengthening. CONCLUSION: The composition and properties of muscle proteins of varying fish species more likely determined protein cross-linking induced by MTGase, thereby affecting their gel properties.

Sugar ester synthesis by thermostable lipase from Streptomyces thermocarboxydus ME168.

The extracellular lipase from Streptomyces thermocarboxydus ME168 was purified to 9.5-fold with 20% yield, following concentration by acetone precipitation, ion exchange chromatography (Resource Q) and gel filtration chromatography (Superdex 200), respectively. The purified enzyme had an apparent molecular mass of 21 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of the lipase was ASDFDDQILG and was different from most other reported lipase. The enzyme showed maximum activity at 50 °C with the half-life of 180 min at 65 °C. It showed high stability at a broad pH range of 5.5-9.5 and was thermostable at the temperature range of 25-60 °C. The K(m) and V(max) were 0.28 mM and 1,428 U/mg, respectively, using p-nitrophenyl palmitate as substrate. It was active toward p-nitrophenyl ester with medium to long acyl chain (C(8)-C(16)). Lipase activity was inhibited by Zn(2+), dithiothreitol (DTT), EDTA and some organic solvents, e.g., ethanol, acetone, dioxane, acetronitrile, tert-butanol and pyridine. Immobilized crude lipase of S. thermocarboxydus ME168 on celite could be used to synthesize sugar esters from glucose and vinyl acetate, vinyl butyrate or vinyl caproate in tert-butanol:pyridine (55:45 v/v) at 45 °C with conversion yields of 93, 67 and 55%, respectively.

Lipids from cephalothorax and hepatopancreas of Pacific white shrimp (Litopenaeus vannamei): compositions and deterioration as affected by iced storage.

Lipids from cephalothorax and hepatopancreas of Pacific white shrimp (Litopenaeus vannamei) stored in ice for up to 6 days were extracted and characterised. The extraction yields of lipids from hepatopancreas (10.65-12.64%) were higher than those from cephalothorax (2.59-2.88%). However, no changes in the extraction yield were observed during the storage (p>0.05). The carotenoid contents of lipids from cephalothorax and hepatopancreas slightly increased within the first 2 and 4 days of iced storage (p<0.05), respectively, but decreased thereafter (p<0.05). With increasing storage time, a progressive formation of hydroperoxide was found as evidenced by the increase in the absorbance band at 3600-3200 cm(-1) in Fourier transform infrared (FTIR) spectra, and increased peroxide values (PVs) (p<0.05). The increases in thiobarbituric acid reactive substances (TBARS), p-anisidine value (AnV) and free fatty acid (FFA) content of lipids were noticeable when iced storage time increased (p<0.05). Those changes indicated that lipid oxidation and hydrolysis occurred in both samples. Phospholipids (PL) were the major components in lipids from cephalothorax (82.51% of total lipids). Nevertheless, lipids from hepatopancreas contained triglyceride (TG) and PL as the dominant components (45.35% and 38.03% of total lipids, respectively). A decrease in the TG content with a concomitant increase in free fatty acid was observed at the end of storage (day 6) (p<0.05). Decreases in unsaturated fatty acids, especially eicosapentaenoic acid (EPA; C20:5(n-3)) and docosahexaenoic acid (DHA; C22:6(n-3)) were noticeable at day 6 of storage (p<0.05). Thus, the extended storage time resulted in the enhanced deterioration of extracted lipids.

Characterization of an unexpected bioemulsifier from spent yeast obtained from Thai traditional liquor distillation.

Crude biopolymer was extracted from spent yeast, lyophilized and fractionated on Sephadex G-100 to yield two fractions coded as fraction I and II. Fraction I was composed of both carbohydrates and proteins, showing emulsifying activity whereas fraction II consisted of only proteins and possessed no activity. Hence composition and chemical characterization of the purified fraction I (bioemulsifier) was analyzed using various analytical techniques. It was found that the sample contained 96% of carbohydrates consisting mainly of glucose with minor quantities of mannose, and 4% of protein built from 17 amino acids with the highest content of serine followed by alanine. The results also indicated that the sample was protein-bound glucan with the average molecular weight of 1.93×10(5) Da. The functional groups and primary structure of the sample were revealed by FT-IR and NMR techniques. The data demonstrate that the sample comprises a mixture of (1→4)-α- and (1→3)-ß-D-glucans bound with protein. Enzymatic hydrolyses using α-amylase and endo 1,3-ß-D-glucanase confirmed the presence of both glucans. Therefore, this bioemulsifier was identified as glucan-protein complex which is different from usual mannoprotein emulsifier derived from yeasts.

Hydroxynitrile lyase from Passiflora edulis: Purification, characteristics and application in asymmetric synthesis of (R)-mandelonitrile.

A hydroxynitrile lyase from leaves of Passiflora edulis (PeHNL) was purified and characterized for the first time. The enzyme is a monomer of 15kDa and 18kDa by SDS-PAGE, and gel filtration, respectively. Asymmetric synthesis of (R)-mandelonitrile from benzaldehyde and acetone cyanohydrin in a biphasic system employing the PeHNL from rinds of P. edulis was carried out. Several parameters influenced the enantiomeric purity of the product and initial velocity of the reaction. Both pH and temperature were important parameters controlling the enantiomeric purity of the product. The optimum pH and temperature were pH 4 and 10°C, respectively. At the optimum pH and temperature, the spontaneous non-enzymatic reaction yielding the racemic mandelonitrile was almost completely suppressed. The PeHNL was stable (more than 80% residual activity after incubation for 12h) in the system of methyl-t-butyl ether (MTBE), dibutyl ether (DBE), hexane (HEX), and diisopropyl ether (DIPE) while diethyl ether (DEE) and ethyl acetate (EA) were not suitable solvents. The initial velocity was markedly affected by the type of organic solvent in the biphasic system, while high enantiomeric purity was obtained when organic solvents having logP lower than 3.5 were used. The highest initial velocity of reaction and enantiomeric purity of (R)-mandelonitrile were obtained in the biphasic system of DBE with the aqueous phase content of 30% (v/v). The optimum substrate concentrations were 250mM for benzaldehyde and 900mM for acetone cyanohydrin, and the optimum enzyme concentration was 26.7units/ml. The highest enantiomeric purity of (R)-mandelonitrile was successfully obtained with conversion and enantiomeric excess of 31.6% and 98.6%, respectively. The enzyme showed considerable reusability in batch reaction with high enantiomeric purity of product. Herein, we reported the characteristics of a unique (R)-PeHNL from leaves of P. edulis. The PeHNL from rinds had been isolated for the first time and the enzyme showed great ability in transcyanation of (R)-mandelonitrile with high e.e. in DBE as the co-organic solvent in a biphasic system.