Effect of media supplements and culture conditions on inulinase production by an actinomycete strain

Streptomyces sp. GNDU 1 produced high levels of extra-cellular inulinase (0.552 IU/ml) after 24 h at pH 7.5, temperature 46 degrees C in the presence of 1% inulin. The optimum temperature and pH for enzyme activity were 60 degrees C and 5.5 respectively. Yeast extract as a nitrogen source was found to be most suitable one for inulinase production whereas ammonium ion was inhibitory to the enzymatic production. All these conditions make Streptomyces sp. GNDU 1, a potential candidate for industrial enzymatic production of fructose from inulin.     

Production of extracellular lipases by Penicillium cyclopium purification and characterization of a partial acylglycerol lipase

Penicillium cyclopium, grown in stationary culture, produces a type I lipase specific for triacylglycerols while, in shaken culture, it produces a type II lipase only active on partial acylglycerols. Lipase II has been purified by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-Sephadex. The enzyme exists in several glycosylated forms of 40-43 kDa, which can be converted to a single protein of 37 kDa by enzymatic deglycosylation. Activity of lipase II is maximal at pH 7.0 and 40 degrees C. The enzyme is stable from pH 4.5 to 7.0. Activity is rapidly lost at temperatures above 50 degrees C. The enzyme specifically hydrolyzes monoacylglycerols and diacylglycerols, especially of medium chain fatty acids. The sequence of the 20 first amino acid residues is similar to the N-terminal region of P. camembertii lipase and partially similar to lipases from Humicola lanuginosa and Aspergillus oryzae, but is different from Penicillium cyclopium lipase I. However, it can be observed that residues of valine and serine at positions 2 and 5 in Penicillium cyclopium lipase II are conserved in Penicillium expansum lipase, of which 16 out of the 20 first amino acid residues are similar to Penicillium cyclopium lipase I.     

一株嗜热子囊菌产生的碱性耐热过氧化氢酶及其应用潜力

研究了一株嗜热子囊菌产过氧化氢酶的摇瓶发酵条件,并对其在纺织工业中的应用潜力进行了评价。以20 g/L糊精和1%(V/V)乙醇为混合碳源时,过氧化氢酶酶活达到1594 u/Ml,比以糊精和乙醇单独为碳源时过氧化氢酶的活力之和还高23%。改变培养基的初始Ph、提高发酵液中的溶氧水平及添加外源过氧化氢,过氧化氢酶的产量进一步提高到2762 u/Ml,比优化前提高了5.8倍。将嗜热子囊菌的过氧化氢酶同来源于牛肝、黑曲霉的过氧化氢酶进行了热(70℃, 80℃, 90℃)、碱(Ph 9.0, Ph 10.0, Ph 11.0)稳定性的比较。结果显示,产自嗜热子囊菌的过氧化氢酶对高温和强碱性的耐受性能明显优于其它来源的酶,在纺织染整工艺中具有良好的应用潜力。     

中温碱性脂肪酶的研究Ⅱ.扩展青霉PF868变株产酶条件

本文报道中温碱性脂肪酶高产变株扩展青霉（Ｐｅｎｉｃｉｌｌｉｕｍ ｅｘｐａｎｓｕｍ）ＰＦ８６８的产酶条件。研究结果表明ＰＦ８６８最适产酶条件为：碳源玉米粉,氮源黄豆饼粉,起始ｐＨ７．５,产酶培养温度２６℃,移种量１０％;亚适量的豆油有利于产酶;７００ｐｐｍ的泡敌不抑制菌丝生长且有利于产酶;适量的非离子表面活性剂Ｔｗｅｅｎ和Ｓｐａｎ类有利于菌丝生长和脂肪酶的释放。     

Production of lipase in a fermentor using a mutant strain of Corynebacterium species: its partial purification and immobilization

Extracellular Corynebacterium lipase was produced using a 2.5 L Chemap fermentor using 1300 ml fermentation medium at temperature 33 degrees C, agitator speed 50 rpm, aeration rate 1 VVM having KLa 16.21 hr(-1). Crude lipase was purified by salting out method followed by dialysis and immobilized using calcium alginate gel matrix followed by glutaraldehyde cross linking Purification process increased specific activity of enzyme from 2.76 to 114.7 IU/mg. Activity of immobilized enzyme was 107.31 IU/mg. Optimum temperature for purified and immobilized enzyme activity were 65 degrees and 50 degrees C respectively. Optimum pH was 8.0 in both the cases, Km and Vmax value for purified lipase were 111.1 micromol/min and 14.7% respectively. Ca2+ (5 mM) was found to be stimulator for enzyme activity. Immobilized lipase retained 68.18% of the original activity when stored for 40 days.     

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.     

脂肪酶产生菌Candida rugosa产酶条件研究

脂肪酶(Lipase,EC3.1.1.3)是用来催化酯类化合物的分解、合成和酯交换的特殊酶,具有高度的化学选择性和立体异构性,它广泛应用于食品、轻纺、皮革、香料、化妆品、洗涤剂、有机合成、医药等领域.本世纪80年代,美国科学家发现酶在近无水的有机溶剂中不仅能保存其催化活力,而且还获得许多新的催化特征[1],此后,脂肪酶在非水相酶催化领域的研究和应用逐渐增多.     

Selection of antagonists of postharvest apple parasites: Penicillium expansum and Botrytis cinerea

The objectives of this study were to constitute a collection of pathogenic agents of economic importance which cause losses of apple fruits after harvest namely Botrytis cinerea and Penicillium expansum and to select in vivo efficient antagonistic strains able to protect fruits against both pathogens at 5 degrees C (P. expansum) and 25 degrees C (P. expansum & B. cinerea). Twenty strains of P. expansum and ten strains of B. cinerea have been isolated from infected apple fruits. Potential antagonistic micro-organisms (thirty three isolates) belonging to yeast, bacteria and fungi have been isolated from apple surface. Six of them (strains Ach1-1, Ach2-1, Ach2-2 belonging to Aureobasidium pullulans (De Bary) Arnaud, and strains 1112-3, 1113-10 and 1113-5 belonging to Aureobasidium pullulans (de Bary) Am. v. pullulans) showed a high level of protection (more than 80%) at 25 degrees C. once inoculated with P. expansum or B. cinerea for 5 days. The highest level of protection against P. expansum (96%) was observed with the application of Ach 2-1. Six days after inoculation of B. cinerea, strains Ach 2-2 and Ach 2-1 insured 100% and 96% of protection, respectively. At lower temperature (5 degrees C), first symptoms of P. expansum appeared 13 days after its inoculation. Percentages of protection observed after apple treatment with one of the six antagonistic strains were ranged from 78% to 94% 20 days after P. expansum inoculation. Strains labelled Ach showed a protective level higher than 90% against this pathogen, followed by strain 1113-10 (90%), strain 1113-5 (89%) and strain 1112-3 (82%). At 26 days post-inoculation, levels of protection decreased but remained higher than 60% (more than 80% with strain Ach2-2 and strain 1113-5, 75% with strain Ach2-1 and 1113-10, 72% with ach1-1, 61% for the other strains). Strain Ach2-2 and 1113-10 were retained as the best antagonists for the subsequent studies.     

链霉菌Z94-2碱性脂肪酶产生条件及酶学性质

在１５２株脂肪酶产生菌中,链霉菌Ｚ９４－２产脂肪酶活力为５９６ｕ／ｍＬ,其最适培养基（ｇ／Ｌ）为：糊精１０、黄豆饼粉３０、尿素１０、Ｋ２ＨＰＯ４０．５、ＭｇＳＯ４０．５、ＮａＣｌ１和ＡＥＯ９０．５,产酶的最适条件为：初始ｐＨ９．５～１０．０,在２６℃培养４８ｈ。用ＰＶＡ橄榄油乳化系统测定该酶的最适ｐＨ９．８,最适温度３７℃,在ｐＨ８．６～１０．２于５℃存放２４ｈ,酶活力不变。０．１４ｍｏｌ／Ｌ的氯     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.     

Optimization of extracellular psychrophilic alkaline lipase produced by marine Pseudomonas sp. (MSI057)

An endosymbiotic Pseudomonas sp. (MSI057), which could produce high yields of lipase, was isolated from marine sponge Dendrilla nigra, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% tributyrin. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 30 degrees C and 9.0, respectively. The enzyme exhibited maximum activity in pH range of 8-9 with an optimum pH 9.0. The activity of purified enzyme was optimum at 37 degrees C and showed 80% activity at 20 degrees C and the enzyme activity decreased dramatically above 50 degrees C. Based on the present findings, the enzyme was characterized as psychrophilic alkaline lipase, which can be developed for industrial applications.     

Characterization of lipases from Staphylococcus aureus and Staphylococcus epidermidis isolated from human facial sebaceous skin

Two staphylococcal lipases were obtained from Staphylococcus epidermidis S2 and Staphylococcus aureus S11 isolated from sebaceous areas on the skin of the human face. The molecular mass of both enzymes was estimated to be 45 kDa by SDS-PAGE. S2 lipase displayed its highest activity in the hydrolysis of olive oil at 32 degrees C and pH 8, whereas S11 lipase showed optimal activity at 31 degrees C and pH 8.5. The S2 lipase showed the property of cold-adaptation, with activation energy of 6.52 kcal/mol. In contrast, S11 lipase's activation energy, at 21 kcal/mol, was more characteristic of mesophilic lipases. S2 lipase was stable up to 45° C and within the pH range from 5 to 9, whereas S11 lipase was stable up to 50 degrees C and from pH 6 to 10. Both enzymes had high activity against tributyrin, waste soybean oil, and fish oil. Sequence analysis of the S2 lipase gene showed an open reading frame of 2,067 bp encoding a signal peptide (35 aa), a pro-peptide (267 aa), and a mature enzyme (386 aa); the S11 lipase gene, at 2,076 bp, also encoded a signal peptide (37 aa), pro-peptide (255 aa), and mature enzyme (399 aa). The two enzymes maintained amino acid sequence identity of 98-99% with other similar staphylococcal lipases. Their microbial origins and biochemical properties may make these staphylococcal lipases isolated from facial sebaceous skin suitable for use as catalysts in the cosmetic, medicinal, food, or detergent industries.     

高通量脂肪酶稳定性测定法-ANS法的建立

根据蛋白质变性后结构变化的特点和荧光探针8-苯胺基-1-萘磺酸 (8-Anilino-1-naphthalenesulfonic acid,ANS) 的特性,建立了一种快捷而准确的脂肪酶热稳定性 (Tm) 测定的新方法——ANS脂肪酶稳定性测定法,即将脂肪酶在25 ℃~65 ℃的不同温度下保温30 min后,加入到ANS反应体系 (0.2 mg/mL酶蛋白,0.05 mmol/L ANS,20 mmol/L Tris-HCl (pH 7.2),100~500 mmol/L NaCl) 中。在激发波长378     

Studies on the enhanced production of extracellular lipase by Staphylococcus epidermidis

Staphylococcus epidermidis isolated from spoiled frozen marine fish samples exhibited optimum lipase activity of 8.1 U within 72 h in batch fermentation. Inducible effect of different sugars, nitrogen sources, salts and metal ions were studied on enzyme production. Trybutyrin induced the enzyme production by twofold. Addition of lactose in the production medium further improved lipase production. Sodium chloride increased lipase production whereas the presence of metals in the media had an inhibitory effect. Cells of immobilized S. epidermidis in agar beads (3%) increased lipase production compared with free cells. The optimum temperature and pH for enzyme activity was 20 degrees C and 7.0 respectively. Lipase retained its 85% stability at pH 6.0 and at 40 degrees C. Immobilized cells with high lipolytic activity and stability may provide commercial advantages over conventional methods of lipase production.     

1,2-dotriacontanedioyl-sn-glycero-3-phosphocholine: A cyclic lipid used for phospholipase A2-catalyzed modulation of the liposome surface charge

An aqueous suspension of liposomes, which were made of cyclic 1, 2-dotriacontanedioyl-sn-glycero-3-phosphocholine (dTPC) and 1, 2-diphytanyl-sn-glycero-3-phosphocholine, was incubatedwith phospholipase A2 (Naja mossambica mossambica) in 0.05 M Tris-HCl, pH 7.7-7.8. At 25 degrees C, approximately 30% of the cyclic lipids was digested into 1-(omega-carboxyhentriacontanoyl)-sn-glycero-3-phosphocholine (lyso-dTPC), the original morphology being preserved but with a decrease in the zeta-potential of the membrane from ca. +2 to -1 mv. At 38 degrees C in 30 min, as much as 45% of the cyclic lipids was attacked by the lipase, which resulted in lowering of the potential to -2.5 to -3 mV. The enzymatic surface-charge modulation was discussed in conjunction with a side-selective attack of the phospholipase on the lipid bilayer.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

Maximizing production of Penicillium cyclopium partial acylglycerol lipase

Penicillium cyclopium partial acylglycerol lipase production was maximized in shaken batch culture. The effect of inoculum size and substrate concentration on the lipase activity released in the culture medium was visualized using a surface response methodology based on a Doehlert experimental design. The main advantage of this approach is the low number of experiments required to construct a predictive model of the experimental domain. Substrate percentage (corn steep, w/v) ranged from 0.1% to 1.9% and inoculum from 100 spores/ml to 3,200 spores/ml. We determined that an optimal set of experimental conditions for high lipase production was 1.0% substrate and 3,200 spores/ml, with initial pH 5.0, temperature 25 degrees C and shaking speed 120 rpm. Between the conditions giving the minimum and the maximum lipase production, we observed a three-fold increase in both the predicted and the measured values.     

Enzymatically enantioselective hydrolysis of prochiral 1,3-diacyloxyglycerol derivatives.

An enzymatically enantioselective ester hydrolysis of prochiral 1,3-diacyloxy-2-substituted-2-propanol to chiral 1-acyloxy-2,3-propanediol was studied. The (R)-monoester was prepared by selection of a suitable lipase and alkyl chain length of the substrate diester. Lipase D from Rhizopus delemer gave (R)-1-isobutyryloxy-2-(2,4-difluorophenyl)-2,3-propanediol with 97%ee and 87% yield at 15 degrees C and pH 5.5. The (R)-monoester is a key intermediate of azole antifungal agents.     

Optimization of conditions for protease production by Chryseobacterium taeanense TKU001

A protease-producing bacterium was isolated and identified as Chryseobacterium taeanense TKU001. An extracellular metalloprotease with novel properties of solvent- and surfactant-stable was purified from the culture supernatant of C. taeanense TKU001 with shrimp shell wastes as the sole carbon/nitrogen source. The optimized condition for protease production was found when the culture was shaken at 37 degrees C for 3 days in 50 mL of medium containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO4.7H2O. Two extracellular proteases (FI and FII) were purified and characterized, and their molecular weights, pH and thermal stabilities were determined. The molecular masses of TKU001 protease FI and FII determined by SDS-PAGE and gel filtration were approximately 41 kDa and 75 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FI were 8, 60 degrees C, pH 6-9, and 60 degrees C, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FII were 7, 60 degrees C, pH 7-9, and 50 degrees C, respectively. TKU001 protease FI and FII were both inhibited completely by EDTA, indicating that the TKU001 protease FI and FII were metalloproteases. TKU001 protease FI and FII retained more than 75% of its original protease activity after preincubation for 10 days at 4 degrees C in the presence of 25% most tested organic solvents. Additionally, the TKU001 protease FI retained 79%, 80%, and 110% of its original activity in the presence of 2% Tween 20, 2% Tween 40, and 2% Triton X-100, respectively. However, at the same condition, the activity of TKU001 protease FII retained 100%, 100%, and 121% of its original activity, respectively. This is the first report of C. taeanense being able to use shrimp shell wastes as the sole carbon/nitrogen source for proteases production. The novelties of the TKU001 protease include its high stability to the solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis.