Production and characterization of a cold-active and n-hexane activated lipase from a newly isolated Serratia grimesii RB06-22

Abstract

A lipase-producing bacterium isolated from raw milk was identified as Serratia grimesii based on 16S rRNA sequence analysis. The extracellular lipase was partially purified by ammonium sulfate precipitation and ultrafiltration. Maximal activity was observed at 10°C, the optimum pH was 8.0 and the enzyme was stable at 5–30°C for 1 h. The K_m and V_max values were 1.7 mM and 0.3 mM/min respectively. It was found that the lipase had the highest hydrolytic activity towards sunflower oil and soybean oil. CaCl₂ had a stimulatory effect on lipase activity, while EDTA and iodoacetic acid slightly inhibited the lipase activity and the enzyme was strongly inhibited by PMSF. The enzyme was compatible with various non-ionic surfactants as well as sodium cholate and saponin. In addition, the enzyme was relatively stable towards oxidizing agents. This lipase exhibited maximum activity in 35% n-hexane retaining about 2191% activity for 1 h.     

长裙竹荪正己烷提取物化学组成及抑菌活性研究

以正己烷为溶剂,对长裙竹荪子实体进行索氏提取,提取率为1.36%。应用GC-MS对提取物的化学成分进行分析,Rxi-1ms柱分离,质谱解析鉴定出55种成分,其中23种成分是首次从竹荪属中检测出来,其主要成分为:羧酸、醇、酮、倍半萜、芳香烃、酯等。提取物对伤寒杆菌、金黄色葡萄球菌、变形杆菌和枯草芽孢杆菌有很好的抑制作用。     

Fatty acid esterification using nylon-immobilized lipase

The esterification of a long-chain fatty acid was conducted using a nylon-immobilized lipase from Candida cylindracea in a nearly anhydrous, nonpolar organic medium, hexane. Butyl laurate was produced from lauric acid and n-butanol at a maximum initial reaction rate of 37 mmol/h. g immobilized enzyme when the substrates were present in equimolar amounts at an initial concentration of 0.5 mol/L. Lower rates were obtained using nonstoichiometric amounts of the substrates. The rate of reaction increased with temperature, reaching a maximum between 35 and 45 degrees C and decreasing sharply at higher temperatures. (c) 1995 John Wiley & Sons, Inc.     

Systematic characterisation and seed oil analysis in candidate plus trees of biodiesel plant, Pongamia pinnata

Candidate plus trees (CPTs) of Pongamia pinnata , a potential biodiesel plant occurring across 10 locations in North Guwahati, were identified based on morphological markers (vegetative and reproductive) using combined analysis over locations. Identified CPTs were then multiplied using seed propagation technique in a nursery bed. The performance of the candidate trees with respect to seed and pod traits, the two most important characters with regard to oil, were evaluated using CROPSTAT software for inferring potential genotypes that can be included in programmes aimed at genetic improvement of the species. Total oil content from the seeds of plus trees was also analysed using solvent extraction procedure at their boiling points. Hexane extraction yielded maximum oil content from seeds (33%) compared with petroleum ether (30%). When the seed to solvent ratio varied, no significant difference was noticed on the total oil yield for an individual tree, although the recovery of solvent and the time taken for oil extraction were significantly reduced at higher ratios of solvent used.     

蜜环菌粉正己烷提取物抗神经炎症研究

蜜环菌是一种药食兼用真菌,其提取物有多种药用价值,本文通过动物体内实验对其抗神经炎症作用进行探究。利用1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）建立了帕金森病小鼠模型,设置空白组、模型组、蜜环菌粉正己烷提取物高低剂量组（10mg/kg、20mg/kg）,连续灌胃给药12d,对小鼠体重、行为学指标、纹状体TH蛋白、Iba-1蛋白、iNOS酶活及相关mRNA表达进行探究。结果发现蜜环菌粉正己烷提取物组能缓解MPTP造模引起的体重减少,对小鼠行为迟缓有显著性改善,增加纹状体TH蛋白表达,抑制Iba-1蛋白过表达,同时对纹状体内炎症因子iNOS及相关mRNA表达有明显的抑制作用。因此,蜜环菌粉正己烷提取物有良好的抗神经炎症活性,可能是通过抑制小胶质细胞的过度激活,抑制iNOS酶活等途径发挥作用。     

Antioxidant,antimicrobial and cytotoxicity potential of n-hexane extract of Cayratia trifolia L

It is of interest to analyze the antioxidant, antimicrobial and cytotoxicity activity of n-hexane extract of Cayratia trifolia L. (C. trifolia). The antimicrobial activity of n-hexane extract of C. trifolia was determined using disc diffusion method against six selected pathogenic microorganisms. The cytotoxicity potential of n-hexane plant extract was also studied against A2780 cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results, n-hexane extract of C. trifolia possess significant antioxidant activity with significant IC50 values in radical scavenging assays. In antimicrobial studies, the maximum zone of inhibition was found in the range of 19.0 ± 0.1 to 22.0 ± 0.1 mm. In MTT assay, inhibition of cell growth with minimal IC50 values of 46.25±0.42μg/mL against A2780 cell lines was observed. Thus, n-hexane extract of C. trifolia is a possible antioxidant, antimicrobial and cytotoxicity agent.     

Is Method 1664A Silica Gel Treated N-Hexane Extractable Material (SGT-HEM: Non-Polar Materials) a Measure of Total Petroleum Hydrocarbons?

Total Petroleum Hydrocarbons (TPH) is a non-specific parameter defined by EPA Method 418.1. The method is simple, inexpensive and straightforward. Freon 113 is used to extract petroleum from the matrix. The Montreal Protocol precludes the production and importation of Freon 113 into the United States. The search for a cost–effective replacement is underway. US EPA method 1664A Silica Gel Treated N-Hexane Extractable Material (SGT-HEM; Non-Polar Materials) has been considered as a replacement for Method 418.1 by some persons. No comparison data has been provided.

The Tosco Corp. Bayway Refinery has a wastewater discharge permit for Petroleum Hydrocarbons (Oil & Grease) that specifies the use of Method 418.1. The intent of this study was to determine if Method 1664A could replace Method 418.1. Method 418.1 is a direct instrumental measurement of the dried extract. Method 1664A is a gravimetric measurement that requires heating the extract to remove the solvent. Also, a key question to be answered is what carbon range petroleum hydrocarbons does heating the extracts lose? Several evaporation procedures and temperatures were studied.

The study showed that the recovery of petroleum hydrocarbons by method 1664A is temperature dependent and that under the study conditions the method 1664A Silica Gel Treated N-Hexane Extractable Material (SGT-HEM; Non-Polar Materials) can be a replacement for method 418.1.     

Biological monitoring of n-hexane exposure in shoe workers

To analyse working conditions and to provide information about the degree to which shoe workers are exposed to n-hexane, the urinary excretion of total 2,5-hexanedione (2,5-HD) was determined in 81 employees in 12 shoe factories. Twenty-five individuals who had experienced no exposure to solvents were used as controls. 2,5-HD was measured in spot urine samples collected from workers at the end of shift. In the urine of shoe workers, the 2,5-HD presented a mean value of 2.33 mg g^-1 creatinine, a median of 1.96 mg g^-1 creatinine. The mean 2,5-HD concentration in the urine samples from non-exposed subjects was 0.28 mg g^-1 creatinine, the median value was 0.18 mg g^-1 creatinine. The mean time-weighted average (TWA) concentration of n-hexane in 12 shoe workshops was 126.1 ppm, ranging from 23 to 215 ppm. We found a significant, but low, correlation (r = 0.40; p < 0.001) between TWA intensity of environmental exposure to n-hexane and the concentration of 2,5-HD in urine. The probable effect of toluene on the concentration of 2,5-HD was also discussed in the present study.     

Identification and characterization of an organic solvent tolerance gene in Helicobacter pylori

BACKGROUND: Pre-cleaning and soaking in glutaraldehyde is the necessary procedure to disinfect endoscopes. However, some chemical-solvent-tolerant bacteria may survive after incomplete endoscopic disinfection. The goal of this study was to identify glutaraldehyde resistance-related genes in Helicobacter pylori. MATERIALS AND METHODS: Lambda-Zap phagemid expression library of H. pylori strain NTUH-C1 was selected with 0.1% glutaraldehyde. The minimal inhibitory concentration (MIC) of glutaraldehyde-resistant DNA fragments of H. pylori NTUH-C1 strain were determined. Imp/OstA recombinant protein was expressed, purified, and used to generate anti-Imp/OstA polyclonal antibody. Imp/ostA knockout, deletion, and complementation strains were constructed. The function of Imp/OstA was monitored by organic solvent tolerance assay, antibiotics susceptibility test, and N-phenylnapthylamine assay. RESULTS: Using Imp/ostA polyclonal antibody against cell lysate of wild-type and imp/ostA mutant showed that it is not essential in H. pylori. Organic solvent tolerance assay demonstrated the role of Imp/ostA in n-hexane tolerance. MIC test showed that the mutation of imp/ostA was susceptible to hydrophobic and beta-lactam antibiotics. NPN assay demonstrated that the level of outer membrane permeability was increased by 50% in mutant strain comparing to wild-type strain (p < .001). CONCLUSIONS: We have identified an Imp/OstA protein that was associated with glutaraldehyde resistance in our clinical strain H. pylori NTUH-C1 by screening of lambda-Zap expression library. Disruption of this protein results in altering membrane permeability, sensitivity to organic solvent, and susceptibility to antibiotics.     

Comparative study of digestive enzymes of squid (Todarodes pacificus) viscera after supercritical carbon dioxide and organic solvent extraction

Three major classes of digestive enzymes of squid viscera were characterized following extraction of oil by supercritical carbon dioxide (SCO₂) and organic solvent, n-hexane. Squid viscera were extracted at temperature, 35∼45°C and pressure, 15∼25 MPa for 2.5 h by SCO₂ with a constant flow rate of 22 g/min. Oil extraction yield increased with the increasing of extraction pressure and temperature. The highest oil extracted residues of squid viscera were used for characterization of digestive enzymes. The activities of protease, lipase, and amylase were highest in n-hexane treated squid viscera samples and lowest in SCO₂ treated samples. The crude extracts of SCO₂ and n-hexane treated squid viscera samples showed almost same optimum pH and pH stability for each of the digestive enzymes. The optimum temperature of protease, lipase, and amylase were found to almost similar in SCO₂ and n-hexane treated samples. But the thermal stability for each digestive enzyme in SCO₂ treated squid viscera were slightly higher than that of n-hexane treated squid viscera. Studies using SDS-PAGE showed no significant differences in protein patterns of the crude extracts of untreated and SCO₂ and n-hexane treated squid viscera indicating no denaturation of proteins.