脂肪酶产生菌的选育及产酶条件的优化

A lipase-producing bacterium strain was isolated from soil and was identified as Pseudomonas sp.. Its lipase yield was improved 2.25-fold by combined beatment of UV irradiation and NTG. The lipase fermentation condition for the mutant strain was optimized with Plackett-Burman design and Response Surface Analysis(RSA), and the formula of the optimum medium suitable for industrial scale fermentation was thereby established. A maximum yield of 87.5 U/ ml was obtained.     

Enhancement of lipase production from hydrocarbons by mutation of Trichosporon fermentans

Lipase high-producing mutants with petroleum products as carbon sources were successfully induced from Trichosporon fermentans WU-C12 by ultraviolet (UV) light irradiation. In the first mutation step, one mutant strain, PU-30, derived from strain WU-C12 was selected. The productivity of extracellular lipase of PU-30 reached 58 units (U)/ml in the medium containing kerosene, being approximately twice the productivity of the parental strain WU-C12. In the second mutation step, the mutant strain 2PU-18 was induced from strain PU-30. In medium containing kerosene, gas oil and liquid paraffin, the 2PU-18 produced 70 U/ml, 62 U/ml and 60 U/ml of extracellular lipase, respectively. When various n-alkanes (C₈-C₁₈) were used as carbon sources, the parental strain WU-C12 produced more than 20 U/ml of lipase only from C₉-C₁₂ alkanes, but 2PU-18 could produce more than 50 U/ml of lipase from C₈-C₁₈ alkanes. When cultivated for 3 days in medium containing liquid paraffin, the activity ratios of extracellular lipase to total lipase and the values of extracellular lipase activity per dry-cell weight were 0.44 and 0.65 U/mg for WU-C12, and 0.62 and 1.82 U/mg for 2PU-18, respectively. These results indicate that the mutant strain 2PU-18 is superior in both total lipase productivity and permeability of lipase to the parental strain WU-C12 when petroleum products are used as carbon sources. Correspondence to: S. Usami     

Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-Directed Mutagenesis

In order to improve the optimum temperature of lipases,the Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique.The recombinant plasmid containing mutant E83 V pPIC3.5K-lip-E83V was expressed in Pichia pastoris GS 115.Comparison experiments of the mutant PEL-E83 V-GS and the wild-type PEL-GS showed that the optimum temperature (45℃) of the mutant was 5℃ higher than that of the wild type.The thermostability of the mutant was similar to that of the wild type.The enzymatic activity of the mutant was 188 U/ml at 37℃,which was 80% that of the wild type in the same conditions.Hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val,and may be responsible for the improvement of the optimum temperature.     

Production, properties and application to nonaqueous enzymatic catalysis of lipase from a newly isolated Pseudomonas strain

A potent bacterium for lipase production was isolated from soil and identified as Pseudomonas species. It produced lipase constitutively. A mutant of this strain with a lipase productivity 3.25-fold higher was obtained by treatment with ultraviolet (UV) and nitrosoguanidine (NTG). Its fermentation condition was optimized to a lipase yield of 87.5 U/ml. The lipase had maximum activity at pH 9.0 and 45 degrees C. It was stable at pHs from 7.0 to 11.0 and below 60 degrees C. The effects of metal ions, surfactants and bile salts were also studied. The lipase was 1,3-specific. In organic solvents, the thermal stability of the lipase was significantly enhanced. Its optimum temperature was also slightly increased. The optimum water activity was found between 0.5 and 0.6. The lipase was successfully applied in organic phase to catalyze the glycerolysis of palm oil for monoglyceride (MG) production, and the enantioselective esterification of (R,S)-2-octanol. The enantioselectivity of the lipase could be enhanced substantially by treatment with an amphipathic.     

Lipase production of Aspergillus oryzae

Aspergillus oryzae produced a small amount of lipase (0.05–0.8 U/wet-g of solid medium) in solid cultures, in contrast to the larger amount (0.46 U/ml) in a shake-flask culture in a modified GYP medium containing 2% glucose, 1% yeast extract and 2% Polypepton. Optimum conditions of lipase production in the submerged culture of A. oryzae were determined in terms of pH, composition of medium, and temperature. In a shake-flask culture at 28°C, the maximum amount of lipase increased to 0.78 U/ml upon the addition of 3% soybean oil to the modified GYP medium. In a jar fermentor culture, 30 U/ml lipase activity was obtained after 72 h at 28°C under appropriate conditions. Lipase production was greatly influenced by the culture temperature, and the optimum temperature for lipase production was about 24°C with a narrow temperature range, which was 10 degrees lower than that for the growth. In the submerged cultures, two kinds of lipase at least exhibiting different substrate specificities were also suggested.     

诱变选育脂肪酶高产菌株及其脂肪酶固定化

以紫外和微波复合诱变选育脂肪酶产生菌 Rhizopus sp. RXF12,获得高产突变株RZ13,其脂肪酶摇瓶发酵单位是出发株的2.62倍。菌株经多次传代,遗传性状稳定。对RZ13菌株的发酵条件进行了正交优化,在25 ℃、pH 8.0的条件下,接入5 %(v/v)的RZ13菌株单孢子悬液 (107个/ml) 振荡培养84 h,达到RZ13菌株最佳产酶状态,脂肪酶活可达95.08 U/ml。考察了脂肪酶性质,在低于40 ℃,pH 7.0~9.0范围内脂肪酶活稳定。经载体筛选及固定化过程优化,选用镁铝水滑石25℃吸附4 h,对RZ13脂肪酶进行了固定化。结果表明,固定化酶的最适作用温度为35~55℃,pH为7.5~9.0,较游离酶的均有较大扩展。     

Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-Directed Mutagenesis

In order to improve the optimum temperature of lipases, the Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique. The recombinant plasmid containing mutant E83V pPIC3.5K-lip-E83V was expressed in Pichia pastoris GS115. Comparison experiments of the mutant PEL-E83V-GS and the wild-type PEL-GS showed that the optimum temperature (45°C) of the mutant was 5°C higher than that of the wild type. The thermostability of the mutant was similar to that of the wild type. The enzymatic activity of the mutant was 188 U/ml at 37°C, which was 80% that of the wild type in the same conditions. Hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val, and may be responsible for the improvement of the optimum temperature. Translated from Microbiology, 2005, 32(1) (in Chinese)     

高产耐热脂肪酶嗜热脂肪地芽孢杆菌的选育

采用氮离子注入技术对耐热脂肪酶产生菌嗜热脂肪地芽孢杆菌（Geobacillus stearothermophilus）L4进行诱变,筛选获得酶活力有较大提高且传代稳定的正突变菌株L4-3;再对L4-3进行紫外线诱变,得到脂肪酶活力提高的正突变菌株L4-3-2,其脂肪酶活力达25．71U／mL,较原始菌株M提高511．9％。高产突变株L4-3-2所产脂肪酶的最适作用温度为50℃,70℃保温60min的剩余酶活为82％,最适作用pH为7．0～8．0,为一种耐热碱性脂肪酶。     

Lipase from marine strain using cooked sunflower oil waste: production optimization and application for hydrolysis and thermodynamic studies

The marine strain Pseudomonas otitidis was isolated to hydrolyze the cooked sunflower oil (CSO) followed by the production of lipase. The optimum culture conditions for the maximum lipase production were determined using Plackett–Burman design and response surface methodology. The maximum lipase production, 1,980 U/ml was achieved at the optimum culture conditions. After purification, an 8.4-fold purity of lipase with specific activity of 5,647 U/mg protein and molecular mass of 39 kDa was obtained. The purified lipase was stable at pH 5.0–9.0 and temperature 30–80 °C. Ca²⁺ and Triton X-100 showed stimulatory effect on the lipase activity. The purified lipase was highly stable in the non-polar solvents. The functional groups of the lipase were determined by Fourier transform-infrared (FT-IR) spectroscopy. The purified lipase showed higher hydrolytic activity towards CSO over the other cooked oil wastes. About 92.3 % of the CSO hydrolysis was observed by the lipase at the optimum time 3 h, pH 7.5 and temperature 35 °C. The hydrolysis of CSO obeyed pseudo first order rate kinetic model. The thermodynamic properties of the lipase hydrolysis were studied using the classical Van’t Hoff equation. The hydrolysis of CSO was confirmed by FT-IR studies.     

产脂肪酶细菌RYXP的诱变选育及突变株产酶条件优化

以"凤丹"牡丹根际土壤中分离筛选到的产脂肪酶菌株Pseudomonas sp. RYXP作为出发菌株,对其进行了紫外线诱变选育,并采用单因素试验和正交试验方法对活性最强正突变株的产脂肪酶基本特性进行了测定。结果表明,出发菌株Pseudomonas sp. RYXP的紫外线诱变最佳条件为:15 W紫外灯30 cm距离照射1 min;将产脂肪酶活性最强的正突变菌株编号为RYXP-3,单因素试验表明RYXP-3产脂肪酶适宜的碳源为玉米淀粉,适宜氮源为豆饼粉,适宜的磷酸二氢钾含量是0.3%,适宜的初始pH值为7;正交试验表明RYXP-3的最佳的产酶培养基成分组成是:玉米淀粉7%,豆饼粉3%,磷酸二氢钾0.3%,初始pH值为8。在优化方案A1B2C2D3产酶条件下,突变株RYXP-3最高的产酶活性达到56.1 U/mL。突变菌株RYXP-3可作为产油牡丹"凤丹"专用促生菌肥开发的备选资源菌株。     

Isolation and screening of Streptomyces sp. Al-Dhabi-49 from the environment of Saudi Arabia with concomitant production of lipase and protease in submerged fermentation

In this study, Streptomyces sp. Al-Dhabi-49 was isolated from the soil sample of Saudi Arabian environment for the simultaneous production of lipase and protease in submerged fermentation. The process parameters were optimized to enhance enzymes production. The production of protease and lipase was found to be maximum after 5 days of incubation (139.2 ± 2.1 U/ml, 253 ± 4.4 U/ml). Proteolytic enzyme increases with the increase in pH up to 9.0 (147.2 ± 3.6 U/ml) and enzyme production depleted significantly at higher pH values. In the case of lipase, production was maximum in the culture medium containing pH 8.0 (166 ± 1.3 U/ml). The maximum production of protease was observed at 40 °C (174 ± 12.1 U/ml) by Streptomyces sp. Lipase activity was found to be optimum at the range of temperatures (30–50 °C) and maximum production was achieved at 35 °C (168 ± 7.8 U/ml). Among the evaluated carbon sources, maltose significantly influenced on protease production (218 ± 12.8 U/ml). Lipase production was maximum when Streptomyces sp. was cultured in the presence of glucose (162 ± 10.8U/ml). Among various concentrations of peptone, 1.0% (w/v) significantly enhanced protease production. The lipase production was very high in the culture medium containing malt extract as nitrogen source (86 ± 10.2 U/ml). Protease production was maximum in the presence of Ca²⁺ as ionic source (212 ± 3.8 U/ml) and lipase production was enhanced by the addition of Mg²⁺ with the fermentation medium (163.7 ± 6.2 U/ml).     

Bile salt-stimulated lipase in non-primate milk: longitudinal variation and lipase characteristics in cat and dog milk

We report the presence of bile salt-stimulated lipase in milk collected from dog and cat. This enzyme has previously been found only in the milk of human and gorilla. Bile salt-stimulated lipase activity in individual dog milk specimens (range: 4.8-107.4 U/ml; 1 U = 1 mumol [3H]oleic acid released/min) was similar, while that in cat milk specimens (range: 2.2-16.9 U/ml) was lower than in human milk (range: 10-80 U/ml). Longitudinal patterns for bile salt-stimulated lipase activity differed depending upon the enzyme source: in dog milk, lipase activity was lowest in colostrum, while in cat milk, lipase activity was highest in colostrum and decreased at mid-lactation. In human milk, bile salt-stimulated lipase activity levels remain fairly constant throughout the first 3 months of lactation. Dog, cat and human milk bile salt-stimulated lipase activity had a neutral-to-alkaline pH optimum of 7.3-8.5, was stable at low pH (above 3.0 for at least 1 h), and was inhibited 95-100% by eserine (at concentrations greater than 0.6 mM). The lipase in the milk of the three species studied had an absolute requirement for primary bile salts (tauro- and glycocholate), and was inhibited by secondary bile salts (tauro- and glycodeoxycholate). These data are the first to report bile salt-stimulated lipase activity in milk from mammals other than the highest primates. Presence of this lipase in non-primate milk will permit the study of the factors that regulate the ontogeny, synthesis and secretion of the enzyme during pregnancy and lactation as well as its function in neonatal fat digestion.     

High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.     

Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution

To expand the functionality of lipase from Rhizopus arrhizus (RAL) we have used error-prone PCR and DNA shuffling methods to create RAL mutants with improved thermostability and the optimum temperature. One desirable mutant with three amino acids substitution was obtained. The mutated lipase was purified and characterized. The optimum temperature of the mutant lipase was higher by 10 °C than that of the wild-type RAL (WT-RAL). In addition, the thermostability characteristic of the mutant was also improved as the result of directed evolution. The half-life (T_1/2) at 50 °C of the mutant exceeded those of WT-RAL by 12-fold. To confirm which substitution contributed to enhance thermostability and the optimum temperature for lipase activity, three chimeric lipases: chimeric lipase 1(CL-1; A9T), chimeric lipase 2 (CL-2; E190V) and chimeric lipase 3 (CL-3; M225I) from the WT-RAL gene were constructed. Each of the chimeric enzymes was purified and characterized. Amino acid substitution at position 190 was determined to be critical for lipase thermostability and the optimum temperature, while the residue at position 9 and 225 had only marginal effect. The mutational effect is interpreted according to a simulated three-dimensional structure for the mutant lipase.     

Production and partial characterisation of extracellular lipase from Aspergillus niger

Summary The production and certain kinetic characteristics of extracellular lipase from Aspergillus niger were investigated. It was possible to substantially enhance the activity of excreted lipase by optimising the interaction between carbon and nitrogen sources applying a two-parameter complete experimental design and response surface analysis. The enzyme was partially purified and a number of kinetic characteristics such as optimum pH and temperature, thermal and pH stability and K_m were determined and discussed. The elevated levels of lipase activity (40.5 U/ml) found in this work competed favourably with most of those reported for lipase hyperproducing fungi.     

Production and purification of a novel extracellular lipase from Alternaria brassicicola

Alternaria brassicicola produced higher quantities (3.2 U/ml) of an inducible extracellular lipase (EC 3.1.1.3) in shaken synthetic medium supplemented with 20 mM methyloleate. After purification, the M r of the lipase was determined as 80 kDa by SDS-PAGE and estimated at 85 kDa using gel filtration, which suggest that the enzyme may be a monomer. The optimum pH and temperature for activity of the enzyme were 9.0 and 25ºC, respectively. Using umbelliferone esters, the lipase was shown highly specific towards a synthetic substrate with long-chain unsaturated fatty acid.     

Enhancement of thermostable lipase production by a genotypically identified extremophilic Bacillus subtilis NS 8 in a continuous bioreactor

Bacillus strain NS 8, a lipase-producing bacterium isolated from a Malaysian hot spring, is able to tolerate a broad range of temperature and pH, which makes it beneficial for this study. It generated PCR products with molecular weight of 1,532 bp, and the 16S rRNA sequence analysis identified it as Bacillus subtilis with accession number AB110598. It showed a 71% similarity index with B. subtilis using Biolog Microstation System. Its lipase production was optimized using a shake flask system by changing the physical (agitation speed, pH and temperature) and nutritional (nitrogen, carbon and minerals) factors. The most suitable combination of the basal medium for lipase production was 2.5% olive oil (carbon), 1.5% peptone (nitrogen), 0.1% MgSO(4) (mineral) at an optimum temperature of 50°C, pH 7.5 and 150 rpm agitation, giving an enzyme yield of 4.23 U/ml. Statistical optimization using response surface methodology was carried out. An optimum lipase production of 5.67 U/ml was achieved when olive oil concentration of 3%, peptone 2%, MgSO(4)·7H(2)O 0.2% and an agitation rate of 200 rpm were combined. Lipase production was further carried out inside a 2-liter bioreactor, which yielded an enzyme activity of 14.5 U/ml after 15 h of incubation.     

Optimization of extracellular lipase production by Geotrichum sp. using factorial design

Response surface methodology was employed to study the effects of carbon source (soy oil, olive oil and glucose) and nitrogen source concentrations (corn steep liquor and NH(4)NO(3)) on the lipase production by Geotrichum sp. The experiment included a 2(4) central composite rotatable design (CCRD) and four others 2(3) CCRD. According to the responses from the experimental designs, the effects of each variable were calculated and the interactions between them were determined. The response surface methodology was applied for the optimization of the nutrient concentrations in the culture medium for the enzyme production, at 30 degrees C. The optimum medium composition for lipase production by Geotrichum sp. was ammonium nitrate 2.1-2.5%, corn steep liquor 13-15% and soy oil 0.6% as carbon source, which lead to a lipase activity of about 20 U/ml. Using olive oil as carbon source, the optimum composition was ammonium nitrate 0.8-1%, corn steep liquor 13-15% and olive oil 0.6%, leading to an activity of 17 U/ml.     

Production of a Pseudomonas lipase in n-alkane substrate and its isolation using an improved ammonium sulfate precipitation technique

Among the various lipidic and non-lipidic substances, normal alkanes within the chain lengths of C-12 to C-20 served as the best carbon substrates for the production of extracellular lipase by Pseudomonas species G6. Maximum lipase production of 25 U/ml of the culture broth was obtained by using n-hexadecane as the sole carbon substrate. The optimum pH of 8 and temperature of 34 + 1 degrees C were demonstrated for the production of lipase in n-hexadecane substrate. The optimum concentration of iron, which played a critical role on the lipase production, was found to be 0.25 mg/l. Lipase production could be enhanced to nearly 2.4-fold by using tributyrin at a concentration of 0.05% (v/v) in the culture medium. High recovery of the lipase protein (83%) from the culture broth was achieved by treating the culture supernatant with Silicone 21 Defoamer followed by ammonium sulfate (60% saturation) fractionation.     

Superinducers for induction of thermostable lipase production by Pseudomonas species NT-163 and other Pseudomonas-like bacteria

Summary Among various additives that affected production of a new thermostable extracellular lipase from Pseudomonas sp. NT-163 , stearyl alcohol was the most effective. Addition of stearyl alcohol (0.5%) brought about ca. 500 fold enhancement of the lipase activity (200 U/ml by p-nitorophenyllaurate method) compared to the case with no additive ( 0.4 U/ml), while olive oil attained only 12–15 U/ml. Palmityl and oleyl alcohols also were highly effective as lipase inducers (150–160 U/ml could be attained). Furthermore, stearyl alcohol induced lipase formation in several other Bacteriol strains 10-times more than olive oil.