脂肪酶产生菌的筛选及不同保藏方法对其产酶活性的影响

从校园食堂附近富含油脂的污泥中分离筛选具有高效脂肪酶活性的菌株。首先以3种不同的平板初筛培养基筛选脂肪酶产生菌,获得的菌株经摇瓶复筛后选出其中一株传代性质稳定,产酶活性高的脂肪酶产生菌,研究其产酶条件及酶学性质,然后分析了斜面法、液体石蜡法、甘油液态冷冻法对此株脂肪酶产生菌的存活率和酶活的影响。实验结果表明,3种平板初筛培养基均能筛选到脂肪酶产生菌,吐温-80琼脂平板培养基筛选效果最佳。经过复筛选定的一株脂肪酶产生菌,最佳产酶条件:发酵液初始p H值9.0,37.0℃培养48 h。酶最适作用温度为38℃,最适作用p H值为9.0,并在p H8.0~10.0之间稳定。液体石蜡法及甘油液态冷冻法在中短期可以使菌种保持较高的存活率和酶活力,为进一步的应用研究奠定了基础。     

除膻菌株的功能性分析

将4株具有除膻效果的霉菌进行功能性分析、包括脂肪酶活力测定试验、生长曲线的测定、产H2S和产生物胺测定试验、抑菌试验、耐盐性和耐亚硝酸盐性试验、拮抗试验与复配试验。结果表明,菌株THZ1、TZH3、TZH4和4QT3的脂肪酶活力分别为4.35、2.34、3.62、2.63 U/mL。4株菌均不产H2S,菌株4QT3产生物胺,其余3株菌不产生物胺。菌株TZH3和TZH4的抑菌能力、耐盐性和耐亚硝酸盐性较好。最终选择TZH3和TZH4进行拮抗试验及复配试验。结果显示,菌株TZH3和THZ4不具有拮抗作用,当TZH3和TZH4的复配体积比为1∶1时,生长情况较优。     

紫外线与微波复合诱变选育高产脂肪酶菌株的研究

研究选育高产脂肪酶菌株。采用紫外线诱变、微波诱变和紫外微波复合诱变出发菌株H3,测定比较酶活,确定最佳诱变效果。结果表明,出发菌株经紫外线和微波诱变后,所产脂肪酶的最高酶活分别为57.984 U/m L和57.1 U/m L,比出发菌株H3提高了38.3%和36.2%,经微波辐射40 s和紫外线照射80 s的复合诱变菌株M3产酶活力最高,达61.6 U/m L,比出发菌株H3提高了46.85%。     

产碱性脂肪酶热带假丝酵母LYSC-3的筛选、鉴定及发酵条件的优化

以橄榄油为唯一碳源,以溴钾酚紫为显色剂,采用琼脂平板法从富含油脂的土壤中筛选到一株产碱性脂肪酶野生型菌株LYSC-3。经形态学、生理生化和分子生物学的鉴定,确定菌株LYSC-3为热带假丝酵母(Candida tropicalis)。经发酵条件优化,菌株LYSC-3的最佳产碱性脂肪酶的发酵条件为:橄榄油10g·L-1、蛋白胨5g·L-1、蔗糖20g·L-1、(NH4)2SO40.5g·L-1、MgSO4·7H2O0.2g·L-1,K2HPO40.2g·L-1,pH8.0,35℃,150r·min-1摇床振荡培养3d,获得碱性脂肪酶最高酶活力达38.6U·mL-1。     

耐有机溶剂脂肪酶产生菌的筛选及其粗酶酶学性质

以体积分数2%苯为筛选压力,利用罗丹明B平板显色法和摇瓶发酵法,从采集的花生地土壤样品中分离筛选得到1株中度耐热、耐碱脂肪酶产生菌,编号为H2。通过形态观察、生理生化特性实验及其16S rDNA基因序列对菌种进行鉴定。结果表明,H2菌株与短小芽孢杆菌(Bacillus pumilus)的亲缘关系最紧密。通过研究得到该菌株的摇瓶发酵条件:产酶培养基为:蛋白胨3%、酵母膏1%、NaCl 0.5%、橄榄油1%,pH7.0,摇瓶发酵温度为28℃,摇床转速为180r/min,发酵周期为48～60h。所产脂肪酶在40℃、pH9.0时酶活性最高,对pH值和温度的适应范围较宽,pH6.0～10.0比较稳定,35～50℃具有较高酶活性。     

产脂肪酶海洋微生物的筛选、鉴定及酶学性质研究

目的:从海洋来源样品中筛选脂肪酶产生菌,并对其进行鉴定和酶学性质研究。方法:以橄榄油为唯一碳源,采用固体平板变色圈观察法筛选脂肪酶产生菌。利用16S r DNA序列分析对其进行鉴定,并对脂肪酶酶学性质进行研究。结果:从海鳗和鲍鱼肠道内容物以及平潭海域、北极和盐湖泥样5个样品中共分离筛选出22株脂肪酶产生菌,其中ZF-16菌株产酶能力较强,根据其形态特征及16S r DNA序列分析鉴定为洋葱伯克霍尔德菌。其酶学性质研究表明,该脂肪酶最适反应p H为9.0,在p H 5.0~10.0范围稳定;最适反应温度75℃,当温度小于40℃时较稳定;低浓度(1 mmol/L)的Na+和K+对脂肪酶的活性有激活作用,Zn2+对脂肪酶有强烈的抑制作用;对C4~C18均有降解作用,尤其对中长链的三辛酸甘油酯(C8)具有较强的降解能力。结论:从海洋来源的样品中筛选到产脂肪酶能力较强的细菌ZF-16菌株,经鉴定为洋葱伯克霍尔德菌。其所产脂肪酶为碱性高温脂肪酶,对C4~C18底物有降解作用,可用于食品烘焙、废纸脱墨和造纸脱胶工业。     

一株酸性脂肪酶高产菌株的筛选与鉴定

目的分离筛选出一株酸性脂肪酶高产菌株并对其进行鉴定。方法通过溴甲酚紫酸碱指示剂进行平板初筛,甘油三丁酸酯透明圈法以及摇瓶培养复筛,筛选获得了一株酸性脂肪酶产生菌株;利用平板透明圈法、橄榄油乳化液滴定法和对硝基苯酚比色法3种方法对所筛菌株进行酶活力测定。结果分离获得一株具有高产酸性脂肪酶活力的菌株,将其命名为菌株WY19。经过检测,其粗酶活力达到11000 U/L。通过对菌株WY19进行形态学鉴定、生理生化实验以及分子生物学鉴定,结合系统发育树分析,确定本研究获得的酸性脂肪酶产生菌为克雷伯氏菌。结论菌株WY19所产脂肪酶为酸性脂肪酶,在食品、医药、油脂加工等领域方面具有较好的应用前景。     

陈醋生高梁糖化菌株的筛选

选择产生淀粉糖化酶较多的3种黑曲霉菌株H303-4-3、AS.3324和CICC2137,对3株菌株产酶条件进行优化使菌株的产糖化酶能力得到提高,经优化后H303-4-3菌株的糖化酶活力最高.在麸皮与豆粕的比例为4:1,麸皮与水分的比例为15:10,pH值5,装瓶量为15g的条件下,接种培养4d的菌株,30℃培养4d,H303-4-3的糖化酶活力达2783.63U/g.     

产酯化脂肪酶微生物的筛选研究

用罗丹明B透明圈法从广东、广西和海南采集的土样中筛选到658株透明圈比较明显的菌株,经发酵测定脂肪酶酶活,获得15株脂肪酶活力超过7U/mL的菌株,采用固定化脂肪酶粗酶制剂的方法进行酯化能力的测定,得到产生酯化脂肪酶能力较强的3个菌株GX-20、GX-35和GX-53,它们的酯化率分别为18.12%、20.65%和19.72%。其中的GX-35菌株经16SrDNA基因序列分析鉴定为Burkholderia cepacia。     

粗状假丝酵母(Candida valida T2)生产脂肪酶的发酵条件

对粗状假丝酵母产生脂肪酶的培养条件进行了研究。结果表明,该菌株产脂肪酶的适宜培养基组成为:桐油15mL/L,黄豆粉30g/L,糊精10g/L,硝酸铵10g/L,MgSO4·7H2O1g/L,K2HPO42g/L,Tween800.5g/L;最佳培养为温度30℃、发酵液起始pH6,摇瓶发酵脂肪酶活力达到1467U/mL。     

毕赤酵母表面展示米黑根毛霉脂肪酶发酵条件

在50 L发酵罐水平对影响毕赤酵母表面展示米黑根毛霉脂肪酶(Rhizomucor miehei lipase,RML)发酵因素进行了研究。最适初始菌体密度(OD600)约为250,最佳诱导pH为6.0;降低诱导温度能有效提高单位甲醇转化率,诱导温度为20℃和25℃时的转化率分别是30℃的1.56倍和1.32倍。在以上最适条件下单位干菌体展示酶活及生产强度分别达275.0 U/g和462.5 U/(L.h),比优化前提高54.5%。研究发现,山梨醇和甲醇混合补料有效地提高了诱导前期(t=12h)的产酶效率,当山梨醇和甲醇混合比例为1∶4及1∶5是以甲醇为单一碳源时酶活力的1.7倍。     

产脂肪酶菌株的筛选鉴定及产酶条件优化

以橄榄油为唯一碳源,采用油脂同化平板从食堂废弃物中筛选出一株产脂肪酶菌株HFE722。通过测定与分析该菌株16S rRNA基因序列,鉴定该菌株为芽孢杆菌（Bacillus sp.）。菌株HFE722在初始条件（发酵温度30 ℃,接种量为1%,自然pH,装液量为100 mL/250 mL,摇床转速为200 r/min）下培养36 h,测得发酵液上清液脂肪酶酶活为2.17 U/mL。优化后所得菌株HFE722产酶的最适发酵条件为：发酵温度30 ℃,发酵周期为36 h,接种量为1%（V/V）,初始pH 7.0,装液量为50 mL/250 mL,摇床转速为160 r/min。在最佳发酵条件下,发酵液上清液酶活可达到5.8 U/mL,酶活较优化前提高了167.28%。     

Optimisation of lipase production by a mutant of Candida antarctica DSM‐3855 using response surface methodology

Response surface methodology (RSM) was used to determine the optimum cultivation conditions for lipase production by a mutant strain of Candida antarctica DSM‐3855. Among four variables, initial pH, soybean meal, and temperature were identified as significant variables for lipase production by full factorial design. A mathematical model was developed to investigate the influences of three factors on lipase activity and to predict their optimum value. According to RSM, the optimum cultivation conditions for the maximum lipase production were found to be: initial pH 6.0, soybean meal 3.38% (w/v), and temperature 26 °C. Under the optimum conditions, the maximum lipase yields of 27.34 U mL^?1 was obtained after 58 h of cultivation in a 15 L fermenter.     

Isolation and characterization of a lipid-degrading bacterium and its application to lipid-containing wastewater treatment

To construct an efficient lipid-containing wastewater treatment system, microorganisms that degrade lipids efficiently were isolated from various environmental sources. Strain DW2-1 showed the highest rate of degradation of 1% (w/v) salad oil among the isolated strains. Strain DW2-1 was identified as Burkholderia sp. and designated Burkholderia sp. DW2-1. The rate of degradation of salad oil, olive oil, sesame oil, and beef tallow by strain DW2-1 were 96.7%, 92.3%, 90.1% and 77.4%, respectively, during a 48-h cultivation. Strain DW2-1 grew well in a synthetic wastewater medium (>1 x 10(10) colony forming unit [CFU]/ml) between 20 degrees C and 38 degrees C, and its rate of degradation of salad oil was above 90% after a 48-h cultivation. The lipase and biosurfactant (BSF) activities of strain DW2-1 after a 48-h cultivation were 1720 U/l and 480 U/ml, respectively. In continuous cultures for lipid-containing wastewater treatment, DW2-1 was stably maintained and degraded more than 90% of salad oil during a 7-d cultivation.     

Astaxanthin preparation by lipase-catalyzed hydrolysis of its esters from Haematococcus pluvialis algal extracts

Five of 8 fungal lipases screened were found to effectively hydrolyze astaxanthin esters from Haematococcus pluvialis algal cell extracts. Among these, an alkaline lipase from Penicillium cyclopium, expressed in Pichia pastoris, had the highest enzymolysis efficiency. Tween80 was shown to be an effective emulsifier in this lipase hydrolysis system for the 1st time. A series of experiments were performed to find optimal conditions for hydrolysis (pH, temperature, reaction time, lipase dosage). In the optimal reaction system, Tween80 and H. pluvialis extracts (mass ratio 1:1) were emulsified and added to the above lipase at a dosage of 4.6 U/μg (relative to total carotenoids), in phosphate buffer (0.1 M, pH 7.0), and incubated at 28 °C for 7 h, with agitation at 180 rpm. The free astaxanthin recovery ratio under these conditions was 63.2%.     

Isolation and identification of lipase producing thermophilic Geobacillus sp. SBS-4S: cloning and characterization of the lipase

A thermophilic microorganism, SBS-4S, was isolated from a hot spring located in Gilgit, Northern Areas of Pakistan. It was found to be an aerobic, gram-positive, rod-shaped, thermophilic bacterium that grew on various sugars, carboxylic acids and hydrocarbons at temperatures between 45°C and 75°C. Complete 16S rRNA gene sequence of the microorganism exhibited homology to various species of genus Geobacillus. A highest homology of 99.8% was found with Geobacillus kaustophilus. A partial (0.7 kbp) chaperonin gene sequence also showed a highest homology of 99.4% to that of G. kaustophilus whereas biochemical characteristics of the microorganism were similar to Geobacillus uzenensis. Based on biochemical characterization, 16S rRNA and chaperonin gene sequences, we identified SBS-4S as a strain of genus Geobacillus. Strain SBS-4S produced several extracellular enzymes including amylase, protease and lipase. The lipase encoding gene was cloned, expressed in Escherichia coli and the gene product was characterized. The recombinant lipase was optimally active at 60°C with stability at wide pH range (6-12). The enzyme activity was enhanced remarkably in the presence of Ca(+2). The K(m) and the V(max) for the hydrolysis of p-nitrophenyl acetate were 3.8mM and 2273 μmol min(-1)mg(-1), respectively. The ability of the recombinant enzyme to be stable at a wide pH range makes it a potential candidate for use in industry.     

Enhanced productivity of electrospun polyvinyl alcohol nanofibrous mats using aqueous N,N-dimethylformamide solution and their application to lipase-immobilizing membrane-shaped catalysts

Electrospun polyvinyl alcohol (PVA) nanofibrous non-woven fabrics have been widely used for cell and enzyme immobilization. Enhancement of the productivity of the material will further enlarge the versatility of them. In this study, a mixture of water and N,N-dimethylformamide (DMF) was used as a solvent of PVA. The productivity defined as ([1 - (amount of polymer which did not come in contact with the collector)/(amount of polymer ejected from the needle for 30 min)] × 100) of electrospun PVA fibers increased from 15 to 92% by increasing the content of DMF from 0 to 10 wt%. As a potential application of the electrospun PVA fibers prepared by the enhanced production system, we encapsulated Burkholderia cepacia (formerly, Pseudomonas cepacia) lipase in the fibers by including lipase powder in the PVA solution before electrospinning, and evaluated catalytic performance of the resultant fibrous catalysts in organic solvent. The lipase encapsulated in the PVA fibers produced from a solution of water and 10 wt% DMF showed a 1.5-fold increase in initial reaction rate in the transesterification of (S)-glycidol and vinyl butyrate to produce (S)-glycidyl butyrate than that encapsulated in the PVA fibers obtained from the solvent without DMF, i.e., water. These findings demonstrate the practicality of the proposed system to enhance the productivity of electrospun PVA fibrous matrices for extended applications of the resultant fibers including the usage as carriers enclosing lipase for reactions in organic solvents.     

假单胞菌JW12脂肪酶的补料分批发酵

研究了假单胞菌ＪＷ１２脂肪酶在２Ｌ和２５Ｌ容积发酵罐的补料分批发酵工艺．通过调整碳源补加速率,控制产酶期发酵液ＰＨ在８．２左右,能有效提高脂肪酶的酶活和表观生产率．在２５Ｌ标准发酵罐中,连续补加吐温－８０,最高脂肪酶酶活为１２９．２μｍｏｌ／（ｍｉｎ·ｍＬ）,表观生产率为１５．９１     

峨眉山野生猕猴桃酿酒酵母筛选研究

从成熟野生猕猴桃自身分离出酿酒酵母菌种,然后与实验室现有优良酿酒酵母菌种一起进行野生猕猴桃酒发酵对比,筛选出MY20酵母菌株为最佳猕猴桃酒酿造菌株,综合菌株在猕猴桃发酵过程中产酸,产酒及其他一些发酵特征比较得出MY20优于其它几种菌株。通过后期驯化,MY20有望成为工业化猕猴桃酒的酿制专用优良菌株。     

乡村自然发酵豆腐乳的真菌多样性研究

为探究乡村豆腐乳内真菌的多样性,本研究以江西上饶市乡村自然发酵豆腐乳为对象,采用稀释平板培养方法,结合形态特征和LSU序列测序进行菌株鉴定、研究自然发酵豆腐乳真菌多样性。结果表明:从上饶市八个采样地24份豆腐乳样品中分离出277株菌株,鉴定出9个属26个种。根据分离频率,青霉属Penicillium（29.04%）、曲霉属Aspergillus（24.65%）、蓝状菌属Talaromyces （22.88%）、镰刀菌属Fusarium（10.57%）是鉴定出所有菌株中的优势属,不同来源地的真菌优势属具有明显差异;多样性指数（H）结果表明,铅山县石塘周边的多样性指数（H=1.92）是八个地区最高,菌群丰富度较高,而德兴市红山生活区的多样性指数（H=0.74）是最低,其菌群丰富度低。相似性指数（Cj）结果表明,铅山县石塘周边与广丰区小康城安利小区中豆腐乳的真菌的相似性系数为1.00,两地菌群相似程度较高,而铅山县紫溪乡文山村与玉山县樟树镇中豆腐乳的真菌相似程度极低,相似性系数为0.29。该研究结果将对提高豆腐乳的品质与安全性提供理论基础。