Lipase from marine Aspergillus awamori BTMFW032: production, partial purification and application in oil effluent treatment

Marine fungus BTMFW032, isolated from seawater and identified as Aspergillus awamori, was observed to produce an extracellular lipase, which could reduce 92% fat and oil content in the effluent laden with oil. In this study, medium for lipase production under submerged fermentation was optimized statistically employing response surface method toward maximal enzyme production. Medium with soyabean meal-0.77% (w/v); (NH(4))(2)SO(4)-0.1m; KH(2)PO(4)-0.05 m; rice bran oil-2% (v/v); CaCl(2)-0.05 m; PEG 6000-0.05% (w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35°C and incubation period-five days were identified as optimal conditions for maximal lipase production. The time course experiment under optimized condition, after statistical modeling, indicated that enzyme production commenced after 36 hours of incubation and reached a maximum after 96 hours (495.0 U/ml), whereas maximal specific activity of enzyme was recorded at 108 hours (1164.63 U/mg protein). After optimization an overall 4.6-fold increase in lipase production was achieved. Partial purification by (NH(4))(2)SO(4) precipitation and ion exchange chromatography resulted in 33.7% final yield. The lipase was noted to have a molecular mass of 90 kDa and optimal activity at pH 7 and 40°C. Results indicated the scope for potential application of this marine fungal lipase in bioremediation.     

离心液相色谱法快速分离纯化神经节苷脂

神经节苷脂在脊椎动物的神经组织细胞膜上含量丰富,除有受体的功能外,还有许多其它功能。采用离心液相色谱法,以国产硅胶Ｇ－６０为填料,分离纯化神经节苷脂,取得了满意的效果。经分离后产物的脂结合唾液酸（ＬＢＳＡ）含量,由１４．３％提高到２４．１％。以蛋白质含量为代表的含氮化合物由２２．０％降至９．６％。回收率为９０．２５％。特点是操作简便、周期短、成本低、效果好。优于ｌａｔｒｏｂｅａｄｓ柱层析法。     

Isolation and partial characterization of latex lectins from three species of the genus Euphorbia (Euphorbiaceae)

Three N-acetylgalactosamine-specific lectins were isolated from the latices of Euphorbia calcina L., Euphorbia dalberi L. and Euphorbia sp. (an undetermined species) by affinity chromatography on fetuin-agarose. They are all glycoproteins [about 12.5% (w/w) carbohydrate] of M, around 140 000 and appear to be tetrameric molecules composed of different subunits. All three lectins have similar amino acid (with high contents of asparagine/aspartic acid, giycine and leucine) and carbohydrate (with glucosamine, mannose.) fucose and xylose) compositions. In addition. they are closely related serologically.     

Purification and characterization of the phosphate/hydroxyl ion antiport protein from rat liver mitochondria.

The phosphate transport protein was purified from rat liver mitochondria by extraction in an 8% (v/v) Triton X-100 buffer followed by adsorption chromatography on hydroxyapatite and Celite. SDS/polyacrylamide-gel electrophoresis (10%, w/v) demonstrated that the purified polypeptide was apparently homogeneous when stained with Coomassie Blue and had a subunit Mr of 34,000. However, lectin overlay analysis of this gel with 125I-labelled concanavalin A demonstrated the presence of several low- and high-Mr glycoprotein contaminants. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer as an additional step in the purification of phosphate transport protein. SDS/polyacrylamide gradient gel electrophoresis (14-20%, w/v) of the hydroxyapatite and Celite eluates revealed one major band of Mr 34,000 when stained with Coomassie Blue. The known thiol group sensitivity of the phosphate transporter was employed to characterize the isolated polypeptide further. Labelling studies with N-[2-3H]ethylmaleimide showed that only the 34,000-Mr band was labelled in both the hydroxyapatite and Celite fractions, when purified from rat liver mitochondria. Further confirmation of its identity has been provided with an antiserum directed against the 34,000-Mr protein. Specific partial inhibition of phosphate uptake, as measured by iso-osmotic swelling in the presence of (NH4)2HPO4, was achieved when mitoplasts (mitochondria minus outer membrane) were incubated with this antiserum. Finally, amino acid analysis of the rat liver mitochondrial phosphate/hydroxyl ion antiport protein indicates that it is similar in composition to the equivalent protein isolated from ox heart.     

Synthesis of poly(A)-containing RNA by isolated spinach chloroplasts.

Chloroplasts were isolated from spinach leaves and the intact chloroplasts separated by centrifugation on gradients of silica sol. Chloroplasts prepared in this way were almost completely free of cytoplasmic rRNA. The purified chloroplasts were incubated with 32PO4 in the light. The nucleic acids were then extracted and the RNA was fractionated into poly(A)-lacking RNA and poly(A)-containing RNA (poly(A)-RNA) via oligo(dT)-cellulose chromatography. The poly(A)-RNA had a mean size of approximately 18--20 S as determined by polyacrylamide gel electrophoresis. The poly(A)-RNA was digested with RNase A and RNase T1, and the resulting poly(A) segments were subjected to electrophoresis on a 10% w/v polyacrylamide gel 98% v/v formamide). Radioactivity was incorporated into both poly(A)-RNA and poly(A)-lacking RNA and into the poly(A) segments themselves. The poly(A) segments were between 10 and 45 residues long and alkaline hydrolysis of poly(A) segments followed by descending paper chromatography showed that they were composed primarily of adenine residues. There was no 32PO4 incorporation into acid-insoluble material in the dark. We conclude that isolated chloroplasts are capable of synthesizing poly(A)-RNA.     

高速逆流色谱法分离凤尾草中的芹菜素和木犀草素

建立了高速逆流色谱分离纯化芹菜素和木犀草素的方法。两相溶剂系统为氯仿-甲醇-水（4：3：2）,上相为固定相,下相为流动相进行洗脱。从100mg粗提物中一步分离得到17．0mg芹菜素和22．5mg木犀草素,经高效液相色谱分析,纯度分别为92％和96％。其化学结构由1H和13CNMR鉴定。     

Isolation,culture and plant regeneration of colt cherry (Prunus avium × pseudocerasus) protoplasts

Large numbers of viable protoplasts were isolated from leaf mesophyll tissues and cell suspension cultures of Colt cherry using 1% (w/v) Onozuka R-10, 0.2% (w/v) Macerozyme R-10, 0.1% (w/v) Driselase, 1% (w/v) polyvinylpirrolidone (av. MW 10 000) (PVP-10) and 2% (w/v) Meicelase, 2% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. Culture media, based on Murashige and Skoog's (MS) salts, supplemented with 9% (w/v) mannitol and various combinations of α-naphthalene acetic acid (NAA), 6-benzylaminopurine (BAP) and zeatin (Z) promoted cell wall regeneration followed by cell colony and callus formation. Protoplasts of both sources were compared in relation to their cultural requirements. Protoplast-derived callus underwent organogenesis.     

高速逆流色谱分离制备甘草中的甘草苷和芒柄花苷

应用高速逆流色谱分离制备甘草中的甘草苷和芒柄花苷。将甘草乙酸乙酯提取物经聚酰胺柱粗分后,30%乙醇洗脱物用高速逆流色谱进一步分离,所用两相溶剂系统为乙酸乙酯-水(5∶5,v/v),转速850 rpm,流速2.0 mL/min,检测波长254 nm,从50 mg30%乙醇洗脱物中得到甘草苷8.7 mg、芒柄花苷4.2 mg,纯度分别为99.5%和97.3%。所得产物的结构经核磁共振谱(NMR)鉴定。利用该方法可以对甘草中的甘草苷和芒柄花苷进行快速的分离和纯化。     

胶束电动色谱法分析奶中两种主要的共轭亚油酸异构体

以胶束电动色谱法对奶样中共轭亚油酸主要的两种异构体进行了分析。在优化条件下(80 mM pH9.0的磷酸盐缓冲液,54 mMSDS,4%(w/v)β-CD,8 M尿素,4%(v/v)乙醇作为运行缓冲液,分离电压25 kV,柱温20℃),胶束电动色谱可在15 min内对奶样中两种主要CLA,即9c,11t-CLA和10t,12c-CLA进行分离测定,最低检出限为0.081 ng/mL。分析结果显示,不同处理奶样中的CLA含量差异显著(P<0.001),但CLA的组成相近,其中的10t,12c-CLA含量差异不显著P=0.999,约为3%;不同品种奶样,如牛奶、水牛奶和羊奶中的CLA含量差异显著(P<0.001),其中CLA含量次序为牛奶>羊奶>水牛奶,并且不同品种奶的9c,11t-CLA与10t,12c-CLA比例差异显著(P<0.05)。     

Affinity chromatography of mustard β-amylase on starch columns

An affinity chromatography method for purification of β-amylase from cytoledons of whit mustard seedlings (Sinapsi alba L.) is described. β-Amylase is bound to starch column, while other contaminating proteins are eluted with the binding buffer. The bound β-amylase is eluted by including dextrin (1%, w/v) in binding buffer. This method yielded a homogenous preparation of β-amylase enzyme, which migrated as a single polypetide band in SDS electrophoresis.     

Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis

An isolate from kimchi, identified as Lactobacillus brevis, accumulated γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in the culture medium. Optimal culture conditions for growth of L. brevis and production of GABA were 6 % (w/v) l-glutamic acid, 4 % (w/v) maltose, 2 % (w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl₂, 2 g Tween 80/l, and 0.02 mM pyridoxal 5′-phosphate at initial pH 5.25 and 37 °C. GABA reached 44.4 g/l after 72 h cultivation with a conversion rate 99.7 %, based on the amount (6 %) of l-glutamic acid added. GABA was purified using ion exchange column chromatography with 70 % recovery and 97 % purity.     

Rapid purification of protein kinase C by high performance liquid chromatography

Protein kinase C was purified from rat brain cytosol by using a high performance liquid chromatography (HPLC), Pharmacia FPLC system. This procedure employed a column chromatography on DE-52, followed by three steps of HPLC procedures with threonine-Sepharose (prepared as described in this report), TSK gel Phenyl-5PW (Toyo Soda), and TSK gel G3000SW (Toyo Soda) columns. Starting from about 30 g of rat brain, approximately 200 micrograms of pure enzyme was obtained. The procedure was very simple and highly reproducible. The enzyme thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of 10% (w/v) glycerol and 0.05% (w/v) Triton X-100, the enzyme could be stored at -80 degrees C for several months.     

Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027

A low-cost medium, MGRS, has been developed for growth and lipase production from Bacillus THL027 at 65 degrees C and pH 7.0. MGRS was composed of 2% (v/v) buffer solution (7.3% (w/v) Na(2)HPO(4), 3.2% (w/v) KH(2)PO(4), pH 7.2), 40 microg ml(-1) FeSO(4) and 40 microg ml(-1) MgSO(4), 0.1% (w/v) (NH(4))(2)SO(4) supplemented with 3% NaCl, 0.1% glucose, 1.0% rice bran oil and 0.5% (w/v) rice bran. The lipase was purified 2.6-fold to apparent homogeneity by ultrafiltration and gel filtration chromatography. Its molecular mass was 69 kDa. The purified enzyme was characterized for its general physical properties.     

Plant regeneration from cotyledon protoplasts of Brassica carinata

Protoplasts isolated from cotyledons of Brassica carinata, underwent sustained division when cultured at 5.0 × 10⁴ ml^-1 in modified 8p medium (KM8P) with 1.0% (w/v) Seaplaque agarose. Cell colonies produced callus when agarose droplets, in which the protoplasts were embedded, were transferred to K8 medium with 0.6% (w/v) Sigma Type I or Type VII agarose at day 16, giving a plating efficiency of 1.6%. Seventy percent of the protoplast derived-tissues produced shoot buds after subculture to MS medium containing 3.0% (w/v) sucrose, 1.125 mgl^-1 BAP, 0.035 mgl^-1 GA and 0.6% (w/v) Type I agarose, resulting in shoot formation from 1.1% of the protoplasts originally plated. Protoplast-derived colonies transferred to hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose produced roots. The latter gave rise to shoots after excision from the parent callus and culture on MS medium with 3.0% sucrose, 0.225 mgl^-1 BAP, and 0.6% (w/v) Type I agarose. Shoots regenerated directly from protoplast-derived calli, or indirectly from roots, developed prolific root systems when placed on hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose.Abbreviations BAP 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - K kinetin - NAA -naphthaleneacetic acid - MES 2(N-morpholino)ethanesulphonic acid, 2,iP-6(,-dimethylallyamino) purine - IAA indole-3-acetic acid - Z zeatin - ZR zeatin riboside     

Temporal accumulation of mannitol and arabitol in Geotrichum candidum

The temporal depletion and accumulation of polyols were investigated in the fungus Geotrichum candidum. The major intracellular polyols were tentatively identified by paper chromatography as mannitol and arabitol. Inositol was also present in small quantities, and trehalose was also detected in appreciable concentrations.Germination and vegetative growth depended on the type and concentration of the sole exogenous carbon source. Mannitol occurred in arthrospores at 9.4% of the dry weight after several days growth in 2% (w/v) glucose solid medium, and became depleted during germination and vegetative growth in liquid medium containing 2% (w/v) glucose, 2% (w/v) sodium acetate or 25% (w/v) glucose as sole carbon source. This hexitol latter accumulated during arthrosporulation. The depletion and accumulation of ethanol-soluble carbohydrate believed to be primarily trehalose was temporally similar to that of mannitol. Arabitol accumulated intracellularly during germination and vegetative growth in sodium acetate medium and 25% glucose medium. This pentitol was not detected intracellularly at any culture age during growth in 2% glucose medium.Prolonged incubation of the culture in 25% glucose medium after stationary phase was reached resulted in the gradual disappearance of arabitol from the arthrospores simultaneously with an increase in intracellular mannitol. In comparison, ethanol-soluble carbohydrate did not change with prolonged incubation in this medium.     

Isolation of thermotolerant,fermentative yeasts growing at 52°C and producing ethanol at 45°C and 50°C

Five thermotolerant, alcohol-producing yeast cultures were isolated from samples obtained from India. Two were identified as ofKluyveromyces marxianus. All five grew on plate-cultures up to 52°C, with maximum growth rates in liquid culture at 40°C. All produced relatively high alcohol concentrations: 5.7 to 7.0% (w/v) at 45°C and 5.0 to 5.5% (w/v) at 50°C when growing on 14.0% (w/v) glucose. All five isolates fermented diluted molasses containing 16.0% (w/v) total sugars, producing 5.6 to 6.0% (w/v) alcohol concentrations. Supplementing the molasses with P, K, Mg and Mn resulted in a 13 to 20% increase in alcohol production at 40°C. The maximum amounts of alcohol produced on supplemented molasses were 7.5 to 8.0 and 6.5 to 7.0% (w/v) at 37°C and 40°C, respectively.     

A rapid preparative method for isolation of neutral and acidic glycosphingolipids by radial thin-layer chromatography

An efficient method to separate neutral and acidic glycosphingolipids (GSLs) from their mixtures within a short period (45-60 min) and with low consumption of solvents (chloroform-methanol-water, 60/35/8 (v/v/v); 250-500 ml) has been developed. This method utilizes a centrifugal thin-layer chromatograph (Chromatotron) and the GSL mixtures (30-400 mg) are applied to glass plates coated with a 1-mm layer of silica gel 60 PF-254. The method (radial thin-layer chromatography) is rapid and simple and the recovery of glycosphingolipids is high (70-80%).     

Screening of Compactin-Resistant Microorganisms Capable of Converting Compactin to Pravastatin

A simple method of using compactin for effective screening of microbial strains with high hydroxylation activity at the 6β position of compactin was developed. Agar plates containing different carbon sources and 500 μg compactin mL⁻¹ were used to screen the microorganisms that can convert compactin to pravastatin. About 100 compactin-resistant strains were isolated from the Basal agar containing 7% (w/v) mannitol as a carbon source, in which two bacteria, Pseudomocardia autotrophica BCRC 12444 and Streptomyces griseolus BCRC 13677, capable of converting compactin to pravastatin with the yield of 20 and 32% (w/w), respectively, were found. High-performance liquid chromatography using C-18 column and two sequential mobile phases, 30% and 50% (v/v) acetonitrile, was also established to simultaneously determine the concentration of compactin and pravastatin in the culture broth. As such, about 2% of target microorganisms could be obtained from the screening program.     

紫茎泽兰9-羰基-10,11-去氢泽兰酮分布积累动态

9-羰基-10,11-去氢泽兰酮为紫茎泽兰（Eupatorium adenophorum）的主要致肝脏毒性成分及杀虫的生物活性成分。从紫茎泽兰叶片中分离提纯得到9-羰基-10,11-去氢泽兰酮（Euptox A）标准品,建立了高效液相色谱法测定紫茎泽兰中Euptox A含量的分析方法。采用C18反相色谱柱,柱温30°C,以甲醇-水（60：40,v/v）为流动相、流速为0.8 mL.min–1、检测波长为255 nm进行测定。Euptox A在紫茎泽兰中的添加回收率为97.3%–103.7%,检测限为0.4μg.g–1。利用建立的方法测定Euptox A在紫茎泽兰体内分布与积累的动态变化规律。结果表明,Euptox A主要分布在紫茎泽兰的叶片中,且在营养生长期积累量高,生殖生长期积累量低。该方法快速、简捷,可用于紫茎泽兰原料及其产品中Euptox A成分的测定。     

Candida galli Strain PGO6: A Novel Isolated Yeast Strain Capable of Transformation of Isoeugenol into Vanillin and Vanillic Acid

Candida galli strain PGO6 isolated from oil-contaminated water is the first isolated yeast strain which is capable to form vanillin and vanillic acid during isoeugenol biotransformation. The products were confirmed by thin-layer chromatography (TLC), changes in the UV absorption pattern and high-performance liquid chromatography (HPLC). The phenotypic and physiochemical characteristics as well as molecular phylogenetic analysis based on amplification the ITS1-5.8S-ITS2 rDNA regions indicated the isolated strain PGO6 was identified as C. galli (GenBank accession number HM641231). Resting cells of C. galli PGO6 from the late-exponential of growth phase were used as biocatalysts for the biotransformation of isoeugenol. The optimal molar conversion of vanillin (48%) and vanillic acid (19%) was obtained after a 30 h incubation using 0.1% (v/v) of isoeugenol and 6 mg of dry weight of cells per ml without further optimization. Under these conditions, the total amount of vanillin and vanillic acid was 583 mg l(-1). Further biotransformation was carried out using 0.5% (v/v) of isoeugenol under the resting cells conditions, yielding a vanillin concentration of 1.12 g l(-1) (molar yield 25.7%) after 60 h incubation. This study brings the first evidence for biotransformation of isoeugenol to vanillin and vanillic acid by a yeast strain.