Purification and characterization of an alkaline lipase from Pseudomonas aeruginosa isolated from putrid mineral cutting oil as component of metalworking fluid

Extracellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa, an extremophile which naturally grows in water-soluble mineral cutting oil (pH 10) used as metalworking fluid (MWF) for cooling and lubrication in industrial metalworking processes. The molecular mass of the purified lipase was estimated by SDS-PAGE to be 54 kDa. The optimum pH and temperature were 11 and 70 degrees C, respectively. The enzyme is stabile over a broad pH range (pH 4-11.5). The lipase preferably acted on triacylglycerols with medium-chain fatty acids. The lipase was inhibited strongly by Zn(2+), Hg(2+), Cu(2+) and slightly by Ca(2+) and Mg(2+). Non-ionic detergents and sodiumdeoxycholate enhanced lipase activity. Alkaline lipase from P. aeruginosa, capable of growing in a water-restricted medium has excellent properties and good potential for biotechnological applications in the metal industry. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool in a water-restricted medium with a variety of applications including organosynthetic reactions and the control and prevention of MWF putrification in the metal industry.     

产耐盐蛋白酶深海细菌的分离鉴定

本文采用高盐富集、复筛发酵及耐盐稳定性实验的方式,从深海海泥中筛选产耐盐蛋白酶的深海海洋细菌。从深海海洋微生物中共筛选出25株耐盐菌,在这25株菌株中有14株在酪蛋白平板上能产生较大的水解圈;通过发酵复筛及耐盐稳定性实验,筛选得到一株酶活性能高且耐盐稳定性好的菌株,将其编号为SWJSS3;对菌株SWJSS3进行16SrDNA的鉴定并初步研究了盐浓度对产酶情况和酶稳定性的影响。菌株SWJSS3在0~10%(m/V)的盐浓度条件下均能生长,在0~10%的盐浓度下,菌液OD600的吸光值范围为0.08-1.98;通过发酵复筛其所产生的蛋白酶酶活在盐浓度为1%时达最高,为233.56±2.16U/mL;所产蛋白酶在终浓度为15%(m/V)的NaCl溶液下,混匀4℃保存1h,残余酶活为初始酶活的40.70±2.06%,继续存放一段时间到第9h,酶活基本保持不变;通过16SrDNA鉴定其为铜绿假单胞菌,与PseudomonasaeruginosaRP2816SrDNA的相似性达到99%以上。     

B.subtilis蛋白酶高产菌株的筛选与鉴定

本文以中性蛋白酶生产菌Ｂ．ｓｕｂｔｉｌｉｓＡＳ１．３９８为出发菌，用ＮＴＧ与紫外辐照相结合的方法对其进行复合诱变，筛选到一株蛋白酶活性明显高于出发菌的正突变株，命名为Ｂ．ｓｕｂｔｉｌｉｓＭＬ。通过对该突变株蛋白酶活性曲线的绘制、酶作用最适ＰＨ的确定、所产一的酶性质的分析及比酶活性的测定，发现该突变株扩酶活性比ＡＳ１．３９８至少高出３０％，最适反应ＰＨ７－８，酶活性受ＥＤＴＡ的强烈抑制，其突变位点很     

玉米半胱氨酸蛋白酶的真核表达及酶学性质表征

玉米半胱氨酸蛋白酶是一种重要的蛋白水解酶,作为C1家族蛋白酶的一员,可在食品、医药等领域广泛应用。本研究从玉米幼苗中提取总RNA,反转录扩增得到玉米半胱氨酸蛋白酶基因,进而构建重组真核表达载体pPICZα-zmCP1,转化毕赤酵母GS115构建重组菌。对表达的玉米半胱氨酸蛋白酶进行酶学性质研究,结果表明该酶最适反应温度为55 ℃,最适反应pH值为6.0,具有较好的热稳定性和有机溶剂抗性,但是pH值的稳定性、金属离子耐受性表现一般。底物特异性研究发现,该酶对小分子底物具有较强的水解能力,且偏爱P1位点为精氨酸的底物。     

高温碱性蛋白酶菌株的选育及酶性质研究

从西藏当雄温泉附近土样中分离到1株高温碱性蛋白酶产生菌株LH 2 2 ,其生长温度范围为4 0～6 5℃,生长pH范围5 0～11 0 ,最适生长pH6 0～8 0 ,经初步鉴定为芽孢杆菌。以LH 2 2为出发菌株,用NTG与紫外线复合诱变的方法,筛选到1株碱性蛋白酶活性显著高于出发菌的正突变株CH 17,其产酶水平比出发株提高4 6 1 2 % ,达到2 380U/mL。该酶的最适作用温度为6 0℃,最适pH值为9 5 ,具有良好的热稳定性,并与去污剂有很好的相容性,酶活性受PMSF的强烈抑制。     

响应面试验优化红花籽油水酶法提取工艺

通过二水平因子分析设计和响应面试验,优化水酶法提取红花籽油工艺。以红花籽油提取率为指标,对酶的种类及添加比例、料液比、总加酶量、酶解时间、酶解温度、酶解p H值进行研究。结果表明:在木聚糖酶UTC-X50、果胶酶NCB3/ZG-040和碱性蛋白酶NCB3/ZG-002比例1∶2∶3(酶活比),总加酶量197.36 U/g,料液比1∶4(g/mL)条件下,先用细胞壁多糖酶(木聚糖酶、果胶酶)在p H 4.2、50℃酶解131 min,再用碱性蛋白酶在p H 9.8、40℃酶解60 min,此工艺条件下红花籽油提取率最高,为84.68%;采用气相色谱法分析脂肪酸组分,发现红花籽油中不饱和脂肪酸相对含量高达91.18%,其中亚油酸相对含量为78.27%,油酸相对含量为12.61%,亚麻酸相对含量为0.10%。     

酶解法提取马油工艺研究

马油具有丰富的药用和食用价值,且开发利用较少,具有研究意义和综合利用价值.采用中性蛋白酶,通过酶解法提取粗马油,运用单因素试验、正交试验法对提取工艺进行优化,获得最佳工艺参数,同时检测粗马油理化性质,并与鱼油等油脂进行对比分析.结果表明,最佳的提取工艺条件为酶解温度55℃、酶解时间2.5h、酶添加量1％、pH值6.5,此时马油提取率为74.74％.研究成果为进一步开展马油精制和脂肪酸成分分析提供了良好的基础.     

比较传统老面中戊糖片球菌的代谢活性与亲缘关系

本研究主要目的探究传统老面中戊糖片球菌的分子特性。8株戊糖片球菌分离自中国中北部不同老面,比较它们发酵葡萄糖产酸的能力,利用脱脂乳分离培养基探究戊糖片球菌产蛋白酶的能力,使用沉淀蛋白法提取胞外分泌蛋白并进行蛋白凝胶电泳分析胞外分泌蛋白,使用Folin-酚法比较不同戊糖片球菌蛋白酶活力。结果表明8株戊糖片球菌均有代谢葡萄糖产酸的能力,其中P.P005能力最佳,经过发酵p H值降低0.41;8株戊糖片球菌均可产生明显水解圈,均具有产蛋白酶的能力;胞外蛋白结果显示P.P002菌株和P.P008菌株分泌蛋白浓度最大,最高酶活力在1057.27～1242.56U/m L之间,在各自最适条件下,产蛋白酶活力从大到小依次是P.P002、P.P001、P.P005、P.P006、P.P003、P.P008、P.P007、P.P004;ERIC-PCR分析亲缘关系分析显示所有的菌株的相似度都在86%以上。本研究为探究传统老面中戊糖片球菌发挥的作用奠定了分子研究的基础。     

Purification and characterization of organic solvent-stable protease from organic solvent-tolerant Pseudomonas aeruginosa PST-01

An organic solvent-stable protease (PST-01 protease) in a culture broth of organic solvent-tolerant Pseudomonas aeruginosa PST-01 was purified by successive hydrophobic interaction chromatography using Butyl-Toyopearl gels. The purified enzyme was homogeneous as determined by SDS-polyacrylamide gel electrophoresis. PST-01 protease had a molecular mass of 38 kDa. The optimum temperature and pH for casein hydrolysis were 55 degrees C and 8.5, respectively. PST-01 protease was stable at pH 8-12 and below 50 degrees C and was determined to be a metalloprotease which was inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon. PST-01 protease inhibited by EDTA was reactivated completely by the addition of zinc or cobalt ions. The stability of PST-01 protease in solutions containing water-soluble organic solvents or alcohols was higher than that in the absence of organic solvent. Furthermore, in general, PST-01 protease was more stable than commercially available proteases, namely, subtilisin Carlsberg, thermolysin, and alpha-chymotrypsin, in the presence of water-soluble organic solvents or alcohols.     

响应面法优化鲣鱼(Katsuwonus pelamis)内脏鱼油酶法提取工艺

为优化蛋白酶酶解鲣鱼内脏条件,提高鲣鱼加工利用率,以鲣鱼内脏为原料,以鱼油提取率为评价指标,采用生物酶解技术提取鲣鱼内脏鱼油,首先对酶进行筛选,其次通过单因素及响应面分析确定pH、酶解时间、酶解温度、液固比、酶添加量等对鲣鱼鱼油提取率的影响,并优化提取工艺。研究结果表明,胰蛋白酶、碱性蛋白酶、胃蛋白酶、木瓜蛋白酶、风味蛋白酶等五种蛋白酶对鲣鱼内脏中鱼油的提取率均有不同程度的提高,其中采用碱性蛋白酶处理的内脏鱼油提取率效果最佳,达到57.46%。此外,鲣鱼内脏中鱼油在pH8.40,酶解时间5.5 h,酶解温度55 ℃,液固比1:1,酶添加量2.0%的条件下鱼油提取率最高,达到58.49%±0.45%。该研究为鲣鱼下脚料的进一步开发和综合利用提供了基础数据和理论依据。     

富血红素多肽产物制备用酶的比较研究

为筛选适于生产富血红素多肽产物的蛋白酶,比较不同蛋白酶对猪血红蛋白的水解效果。在AS1398 中性蛋白酶、2709 碱性蛋白酶、木瓜蛋白酶和胰蛋白酶的最适水解pH 值及温度下,以L16(45)正交试验研究酶种类、酶质量分数、底物质量分数和水解时间等因素对水解率、水解液中粗多肽含量和血红素含量等指标的影响。结果表明：酶种类和水解时间两因素对水解率指标的影响达到极显著水平(P ＜ 0.01);底物质量分数是影响粗多肽含量指标的最重要因素;酶种类对血红素含量指标的影响则达到显著性水平(P ＜ 0.05)。中性蛋白酶有利于提高水解率,木瓜蛋白酶有利于提高水解物的粗多肽含量,而碱性蛋白酶则有利于提高水解物的血红素含量。     

海洋细菌Pseudoalteromonas flavipulchra HH407低温碱性蛋白酶的发酵条件和酶学性质研究

对一株分离自连云港海域的海洋细菌(Pseudoalteromonas flavipulchra HH407)菌株产蛋白酶的条件及酶学性质进行研究。结果表明,在发酵温度为20℃、培养基pH 值为8.0、NaCl 质量浓度为20g/L、接种量2.5% 和装液量20% 时产酶较高。甘露醇、果糖、乳糖、蔗糖、麦芽糖、鱼粉和酵母粉有利于蛋白酶的产生。该酶的最适作用温度为35℃,0℃时相对酶活力达21.7%。45℃条件下保温60min 后酶活力仍能保持80% 以上。酶的最适作用pH 值为10.0,pH 值为7.0～11.0 范围为时,酶活力相对比较高。酶在pH8.0～11.0 范围内稳定,最稳定的pH 值为10.0,属于低温碱性蛋白酶,Mn2+、Cu2+、Ca2+ 对其具有较强的激活作用,Hg2+ 较强地抑制其酶活力。苯甲基磺酰氟(PMSF)能完全抑制该蛋白酶,EDTA 无影响表明该酶属于丝氨酸蛋白酶。     

PURIFICATION OF A PROTEASE FROM AN ALKALOPHlLIC BACILLUS SUBTILIS CHZ1 ISOLATED FROM A ZIMBABWEAN HOT SPRING

A proteolytic enzyme produced by Bacillus subtilis CHZ1 was purified using ammonium sulfate precipitation, gel filtration and cationic exchange on S‐Sepharose fast flow column chromatography. Production of the protease was higher when the Bacillus strain was cultured in a synthetic medium, M162, supplemented with 0.3% (w/v) organic compared to inorganic nitrogen sources. Enzyme production was growth dependent and production was highest when tryptone was used as the nitrogen source. When run on SDS‐PAGE gel, the purified enzyme gave a 35 kDa band, suggesting that it consisted of one polypeptide chain. High enzyme activity was observed in the pH range of 6–10 with a maximum value at pH 8.0 when 0.5% (w/v) azocasein was used as the substrate. Optimum temperature for protease activity was found to be 60–80C, and the enzyme had considerable thermal stability for 5.5 h retaining about 90% activity after 5.5 h. At 2.5 mM concentration, PMSF, Ag⁺ and Hg⁺ inhibited activity of the protease. Metal cofactors like Mn²⁺, Mg²⁺ and Fe²⁺ increased the enzyme activity by about 20%. Zn²⁺, Cu²⁺ and Ca²⁺ did not affect the enzyme's activity. The pH and thermal stability as well as high specific activity of this enzyme can be exploited for industrial applications.     

白曲酸性蛋白酶在无孢黑曲SH-2中的克隆表达及酶学性质分析

白曲霉酸性蛋白酶在白酒酿造发酵过程中具有溶解发酵原料的颗粒,促进微生物繁殖,分解蛋白质生成香味物质,降解酵母菌体蛋白等多种功能,可以提高白酒风味,因此被广泛应用于白酒生产中。本研究利用经基因工程改造的低内源蛋白背景的黑曲霉宿主SH-2,表达白曲霉酸性蛋白酶基因pepB。通过PCR技术获得pepB以及表达元件糖化酶启动子PglaA、糖化酶终止子TglaA、乳清酸核苷-5-磷酸脱羧酶标记基因pyrG,在pMD18-T载体基础上,构建了pepB表达载体,通过PEG介导转化法转化无孢黑曲酶SH-2。重组菌株经发酵罐发酵240 h,发酵粗酶液酶活达9722 U/mL,是报道的白曲原始菌株酶活的8.5倍,SDS-PAGE结果显示表达产物分子量约为47 ku。酶学性质分析结果表明,该酸性蛋白酶最适反应温度为35℃,最适pH为4.0,Mn~(2+)、Cu~(2+)对酶活有显著地激活作用。最后,探究了在不同发酵初始pH下重组菌株酶活的变化,结果显示,在pH 4.5~6.5范围内,适当提高发酵初始pH,酸性蛋白酶酶活会提高。以上结果表明,本研究成功构建了一株能胞内高效表达白曲酸性蛋白酶的菌株。     

高产蛋白酶菌株的分离、鉴定及酶学性质研究

该研究旨在从非传统环境（磷矿）中分离、筛选高产胞外蛋白酶菌株，对菌株进行形态学观察、生理生化和分子生物学鉴定，并研究其蛋白酶的酶学性质。结果表明，分离筛选到一株蛋白酶高产菌株，编号为PB5，该菌株被鉴定为贝莱斯芽孢杆菌（Bacillus velezensis），该菌株产胞外蛋白酶活力为562.3 U/mL，胞外蛋白酶的最适pH值为10，在pH 7～10有较好的pH稳定性；最适温度为60 ℃，20～50 ℃时有较好的稳定性；Na+、Mg2+、Mn2+、K+对蛋白酶活力有明显的促进作用；Fe2+、Ag+、乙二胺四乙酸（EDTA）、苯甲基磺酰氟（PMSF）、十二烷基硫酸钠（SDS）对蛋白酶活力有明显抑制效果。     

植物乳杆菌DL3对草鱼片中腐败菌的抑制

应用植物乳杆菌(Lactobacillus plantarum)DL3对草鱼片中铜绿假单胞菌和腐败希瓦氏菌进行抑制作用研究,从感官评价、微生物指标和理化指标验证其抑菌保鲜效果。结果表明:4℃冷藏条件下,菌株DL3处理组能有效维持草鱼片的感官品质。与对照组相比,菌株DL3可使草鱼片中铜绿假单胞菌和腐败希瓦氏菌的细菌数量分别降低2.06和0.97 lg CFU/g,能有效抑制草鱼片中腐败菌的生长繁殖。鱼片中pH呈现先降低后逐渐增加的"V"型变化趋势,表明添加菌株DL3对pH上升具有延缓作用。菌株DL3单独处理组的TVB-N值为18 mg/100 g,能够有效控制蛋白质的降解。研究表明菌株DL3可作为淡水鱼及其制品的生物保鲜剂,对控制水产品腐败具有一定的应用价值。     

Isolation of the Pseudomonas aeruginosa gene affecting uptake of dibenzothiophene in n-tetradecane

The dsz desulfurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred into the chromosomes of Pseudomonas aeruginosa strain NCIMB9571 using a transposon vector. All of the recombinant strains completely desulfurized 1 mM dibenzothiophene (DBT) in n-tetradecane (n-TD) except one, named strain PARMI. Strain PARMI was unable to desulfurize DBT in n-TD, but was able to desulfurize it in water. The n-alkane utilization ability, the biosurfactant production and the fatty acid composition of cells in strain PARMI were the same level as those of the other recombinants. The transposon insertion area of strain PARMI was analyzed by transposon tagging. We cloned three possible open reading frames (ORFs), designated hcuA, hcuB and hcuC, from the genomic DNA of P. aeruginosa NCIMB9571 using the transposon insertion area of strain PARMI as a DNA probe. Examination of the sequence revealed the transposon was inserted into hcuA. The full length of the hcuABC genes transformed into strain PARMI achieved 87% recovery of the desulfurization activity of DBT in n-TD, but partial hcuABC genes achieved only 0-12%. These results indicate that DBT desulfurization in the oil phase by recombinant P. aeruginosa strain NCIMB9571 requires the full length of the hcuABC gene cluster. The hcuABC gene cluster relates to DBT uptake from the oil phase to inside of the cell, and the uptake ability is independent of the n-alkane utilization ability, the biosurfactant production and the fatty acid composition of cells.     

高产碱性蛋白酶低温菌的筛选及发酵

从123株南极低温细菌中筛选到1株高产低温蛋白酶的菌株,16S rDNA系统发育分析鉴定为Pseudoalteromonas。经生长条件优化,羧甲基纤维素钠和蛋白胨分别为发酵培养的最适碳源和氮源,该菌在初始pH7.0～8.0、10℃培养5d后产酶酶活达0.48U/mL,约为优化前(0.27U/mL)的1.8倍。对粗酶液进行酶学性质初步研究,此酶在pH9.0、30℃下活性最高,是一种低温碱性蛋白酶,在工业领域有很大的应用潜力。     

Lipase production by solid fermentation of soybean meal with different supplements

The aim of this work was to study the production of extracellular lipases by solid state fermentation in soybean meal with different supplements. Lipase production by two microorganisms, screened by their potential for lipase production, was followed in terms of hydrolytic activity at different pH values(4, 7 and 9). The supplementation of the medium with urea and soybean oil significantly increased the enzyme production for all studied pH and microorganisms. Microorganism Penicillium P58 and P74 showed the possibility of production of different lipases with alkaline and acidic characteristics. In soybean meal supplemented with urea and soybean oil, this microorganism yielded 139.2 and 140.7U lipase/g of dry substrate in 48 h of fermentation, in alkaline and acidic conditions, respectively. Different behavior was observed when the enzyme extract was evaluated in neutral conditions, which yielded 180.0U lipase/g in 72 h. A new promising lipase producer strain was testedon soybean meal with different supplements. The strain produced a lipase with high activities by solid state fermentation of soybean meal supplemented with urea and soybean oil.     

High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation

The methylotrophic yeast, Pichia pastoris, is widely used as a host strain for the production of a variety of heterologous proteins. We used P. pastoris for the production of recombinant human serum albumin (rHSA). In several runs of fed-batch fermentation, rapid degradation of rHSA was observed, coinciding with a sudden increase of protease activity in the culture broth. Monitoring the changes in the concentration of the medium components during fermentation suggested that this phenomenon was caused by nitrogen starvation. Increased initial concentrations of ammonia and phosphoric acid in the medium prevented the protease production during fermentation. Using this improved medium, stable production of rHSA of around 1.4 g/l was achieved. Although protease activity in the culture broth of the improved medium was not detected by the casein plate method at the end of fermentation, potential protease activity remained and could be activated by decreasing the pH of the culture broth, a high degradation rate of 660 mg HSA/l/h was observed at pH 4.3, but degradation did not occur above pH 5.9.