南极产低温脂肪酶菌株Psychrobacter sp. 7195 的 选育、发酵条件及酶学性质研究

从南极普里兹湾深海沉积物中筛选到一株产低温脂肪酶的菌株7195,细菌学形态鉴定及16S rDNA序列分析表明该菌株属于嗜冷杆菌属 (Psychrobacter). 生长特性研究表明该菌株属于耐冷菌,其最适生长温度范围为5～15°C, 7195菌株能利用多种碳、氮源产酶.粗酶液经硫酸铵盐析、DEAE cellulose-52 柱层析进行初步分离纯化后进行酶学性质的研究. 该菌株所分泌的脂肪酶最适作用温度为30°C,最适pH值为9.0,对热敏感,60°C热处理10min剩余酶活为30%,是典型的低温酶. Ca2+、Mn2+、Cu2+对该酶有较为明显的激活作用,而Co2+、Zn2+、Hg2+、Rb2+、Cd2+、Fe3+、EDTA则能抑制酶活,此外该脂肪酶能在高浓度的SDS、CHAPS、Triton X-100、Tween 80、Tween20等变性剂中表现出较好的稳定性.     

低温耐热脂肪酶的筛选、纯化及特性研究

目的:对一株低温耐热脂肪酶产生菌Pseudomonas RT-7进行产酶、纯化和特性研究.方法和结果:该菌的发酵液经50%硫铵沉淀、DEAE-Sepharose及Sephacryl S-100分离获得了纯化的脂肪酶(PL-7).SDS-PAGE电泳估算其表观分子量为44kDa,对底物特异性、作用温度、作用pH和耐热性的研究表明该酶为碱性脂肪酶,最适温度在15～20℃.该酶对C≤12链长的甘油三酯有较好的水解能力.该酶具有在低温和高温下稳定而在中温下不稳定的特点:表现为该酶经60℃30min处理后残余酶活高达93.33%,90℃处理30min后残余酶活仍有35.19%,而在40℃处理30min酶活仅残余28.23%.结论:该酶为低温碱性脂肪酶并在高温条件下具有很好的耐热性.     

交替假单胞菌(Pseudoalteromonas sp.)DY3菌株产内切葡聚糖酶的性质研究及基因克隆

从深海样品ES0109中分离到一株具有高内切葡聚糖酶活力的细菌DY3, 16ＳrDNA序列分析表明该菌与交替假单胞菌属(Pseudoalteromonas sp.)的Pseudoalteromonas citrea和Pseudoalteromonas elyakovii的同源性为99%。PCR扩增DY3的内切葡聚糖酶基因cel-X全长1479bp,编码一个492AA的蛋白质。酶的氨基酸序列分析表明CelX与Pseudoalteromonas haloplanktis的内切葡聚糖酶CelG有95%的相似性,包括一个糖基水解酶家族5的催化结构域,一个连接序列和位于C端的的CBM5结构域。对酶性质的初步研究发现,CelX的最适温度为40 ℃,酶的最适pH在6～7之间。     

产脂肪酶嗜碱细菌的筛选及酶学性质研究

目的:筛选产脂肪酶嗜碱细菌,并研究其酶学性质.方法:以豆油为唯一碳源的固体平板筛选产酶菌株,16S rDNA同源性分析确定微生物菌属,单因素实验优化产酶条件、研究酶学性质.结果:筛选出1株产脂肪酶的嗜碱菌株,鉴定为假单胞菌(Pseudomonas),命名为Pseudomonas sp.C-36.菌株产酶的最佳培养条件为:甘油2%(V/V).蛋白胨0.7%(W/V),酵母提取物0.5%(W/V),K2HPO4 0.2%(W/V),MsS04·7H2O 0.05%(W/V),NaCl 0.3%(W/V),Triton X-100 0.01%(W/V),pH 9.5,转速180r/min,37℃下培养24h,产酶量为2.782 IU/ml.该酶的最适温度和pH分别为40℃和9.0,50℃时酶活半衰期为2h.Ca2+等金属离子对该酶酶活具有促进作用,而Zn2+对酶活的抑制作用明显.有机溶剂的耐受性实验表明,该酶在疏水性有机溶剂中稳定性良好.结论:筛选得到1株嗜碱的脂肪酶产生菌C-36,并鉴定为Pseudomonas.     

南极深海沉积物宏基因组DNA中低温脂肪酶基因的克隆、表达及性质分析

从南极普利兹湾深海900米深的沉积物中提取获得宏基因组DNA,并通过设计引物,从中克隆到全长为948bp的低温脂肪酶(lip3)开放阅读框完整序列,该基因编码一个由315个氨基酸残基组成、预计分子质量为34.557ku酶蛋白(Lip3);氨基酸序列上的GFGNS(GXGXS)和G-N-S-M-G(GXSXG)在许多脂肪酶中有很高的保守性,它们是水解机制所必需的序列,也是丝氨酸水解酶中最保守的序列.构建了lip3基因重组表达载体,并在大肠杆菌中获得表达,采用镍离子亲和层析柱对表达的酶蛋白Lip3进行纯化,得到约35ku蛋白条带,酶学性质的分析表明,该酶的最适作用温度为25℃,在0℃时表现为最高活力的22%,最适pH值为8.0,对热敏感,35℃热处理60min剩余酶活为10%,以硝基苯棕榈酸酯为底物,Lip3的酶促反应常数Km值随着反应温度的升高而升高,是典型的低温酶.     

基于核糖体基因序列快速鉴定产脂肪酶微生物

利用含罗丹明B的橄榄油检测平板从中国各省市油污土壤中分离、筛选产脂肪酶微生物菌株,扩增细菌的核糖体基因16S rDNA序列和真菌的ITS2序列,分析核糖体基因簇DNA,并结合形态学特征从而对产脂肪酶菌株进行分子生物学鉴定.核糖体基因16S rDNA序列分析及系统发育分析表明,分离得到的产脂肪酶细菌分别属于枯草芽孢杆菌(Bacillus subtilis)、产碱假单胞菌(Pseudomonas alcaligenes)、洋葱伯克霍尔德氏(Burkholderia cepacia)、琼氏不动杆菌(Acinetobater jurii)、嗜麦芽窄食单孢菌(Stenotrophomonas maltophilia)和荧光假单胞菌(Pseudomonas sp.);真菌核糖体基因转录间隔区(ITS2)序列及同源性分析表明产脂肪酶真菌分别属于黑曲酶(Aspergillus niger)、白地酶(Galactomyces geotrichum)、解脂耶氏酵母(Yarrowia lipolytica)、丝孢酵母(Trichosporon guehoae)和假丝酵母(Candida sp.).研究结果表明,核糖体基因簇的DNA分析技术为从自然界分离、鉴定产脂肪酶菌种提供了一种快速有效的手段,为产脂肪酶微生物资源开发利用奠定了技术基础.     

思茅松毛虫肠道产脂肪酶菌株的筛选鉴定及酶学性质初步研究

利用纯培养和筛选培养,从思茅松毛虫幼虫肠道中分离得到7株产脂肪酶的菌株.通过提取基因组DNA并进行16S rDNA序列测定,构建产酶菌株的系统发育树,初步鉴定结果显示:菌株D2、D12、D19属于假单胞菌属(Pseudomonas sp.),菌株D7、D17属于芽胞杆菌属(Bacillus sp.),菌株D9、D16属于克雷伯氏菌(Klebsiella sp.).初步研究所产脂肪酶的酶学性质,确定这些酶的最适作用温度30～40℃、最适作用pH值8.0～9.0,为中温碱性脂肪酶.     

嗜热毛壳菌热稳定脂肪酶基因(Im)的克隆及其在毕赤酵母中的表达

研究从嗜热毛壳菌Chaetomium thermophilum中克隆了一个新的脂肪酶基因(lm).其中DNA序列包含一个由870个碱基构成的开放阅读框,编码289个氨基酸,含有4个内含子,没有信号肽序列.序列提交GenBank,登录号为GU338248.将该基因在毕赤酵母中表达.在甲醇的诱导下,重组蛋白得到了高效表达,第6天的表达量最高,蛋白达到0.428mg/mL,酶活力为19.77U/mg.SDS-PAGE检测该蛋白的分子量为35kDa.该脂肪酶的最适反应温度为60℃,具有热稳定性,在40-80℃热稳定,80℃处理60min仍有65％的相对酶活.该酶最适反应pH值为10.0,在pH 9.0--12.0酶活相对稳定.该酶具有较好的热稳定性和耐碱性,具有良好的工业应用价值.     

一株耐冷玫红假裸囊菌HD1031的鉴定及其所产纤维素酶的研究

[目的]以纤维素为唯一碳源,从四川省阿坝自治州黄龙沟的高山低温环境中分离筛选产纤维素酶的耐冷菌,并研究菌株的产酶特征.[方法]根据菌株的ITS序列分析及形态特征,对菌株进行鉴定.利用DNS法测定纤维素酶酶活性.[结果]从四川省阿坝自治州黄龙沟的高山腐殖土中筛选出一株产纤维素酶的耐冷菌HD1031,经鉴定该菌为玫红假裸囊菌(Pseudogymnoascus roseus).该菌可在4℃-25℃生长,最适生长温度为16℃-17℃.该菌在以微晶纤维素和玉米芯粉为碳源、硫酸铵和Tryptone为氮源的培养基中,17℃、160 r/min摇瓶发酵8d后产生纤维素酶,其中内切葡聚糖酶酶活为366.67 U/mL,滤纸酶酶活87.6 U/mL,β-葡萄糖苷酶酶活90.8 U/mL,酶最适反应pH为6.0,最适反应温度为50℃.[结论]筛选获得一株产纤维素酶的耐冷菌HD1031,此菌株所产纤维素酶在20℃-40℃下活性较高,对热敏感,具有低温纤维素酶的特点.     

高产耐高温脂肪酶生产菌的筛选与鉴定

从小笼包蒸屉垫中筛选得到了两株脂肪酶高产菌株J2和J3,经形态观察以及26S rRNA基因(26S rDNA)序列比对鉴定,两株菌分别属于Aureobasidium属的两个变体。200 r/min、30℃下摇瓶发酵3-5 d后,以对硝基苯酚棕榈酸酯(p-NPP)作为底物,用分光光度法测得J2和J3发酵上清液中的脂肪酶酶活分别为10.61 U/m L和14.43 U/m L。对两株菌所产脂肪酶的耐热特性研究显示,菌株J2产脂肪酶的最适反应温度为50℃,并且酶液在50℃保温5 h无酶活损失;另一株菌J3所产脂肪酶的最适反应温度为60℃,酶液在50℃保温5 h后酶活剩余42.19%,在40℃保温5 h没有酶活损失。这表明J2和J3菌株所产脂肪酶具有较好的热稳定性和较高的最适反应温度。     

Molecular Cloning and Expression of a Cold-Adapted Lipase Gene from an Antarctic Deep Sea Psychrotrophic Bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. 7323

A psychrotrophic bacterium producing a cold-adapted lipase was isolated from the deep-sea sediment of Prydz Bay, Antarctic and identified as a Pseudomonas strain. Determination of the nucleotide sequence of the gene encoding a lipase from Pseudomonas sp. 7323 (lipA) revealed that LipA is composed of 617 amino acid residues with a calculated molecular weight of 64,466 Da. LipA has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine. The lipase purified from the Escherichia coli transformant (rLipA) by metal-chelating chromatography exhibited the same electrophoretic mobility as did the wild-type lipase (wLipA) purified from strain 7323, and both enzymes were quite similar in physicochemical properties. The optimal temperature and pH value for the lipases activity were 30 degrees C and 9.0, respectively. They were unstable at temperatures above 25 degrees C and only retained half of their highest activity after incubation at 60 degrees C for 5 min. These results indicated that the enzymes were typical alkaline cold-adapted enzymes. Both enzymes were particularly activated by Ca(2+). Additionally, the enzymes hydrolyzed p-nitrophenyl caprate and tributyrin at the highest velocity among the other p-nitrophenyl esters and triglycerides.     

Low-temperature lipase from psychrotrophic Pseudomonas sp. strain KB700A

We have previously reported that a psychrotrophic bacterium, Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at -5 degrees C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca2+. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca2+-dependent lipase. Addition of Mn2+ and Sr2+ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca2+. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C10 acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35 degrees C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.     

南极交替单胞菌R11-5产卡拉胶酶的发酵条件优化

【背景】极地寒冷环境中发现了大量具有潜在应用前景的冷适应酶,同时也存在种类繁多的海藻多糖降解菌,因此极端环境微生物是筛选获得新颖、高效多糖降解酶的重要新源泉。由于筛选培养基通常并非野生菌发酵产酶的最优条件,为了使野生菌的产酶效率达到最高,需要对其培养条件进行优化,从而为其深入研究及开发利用提供依据。【目的】对一株产卡拉胶酶的南极菌株进行种属鉴定,并采用响应面法对该菌的发酵产酶条件进行优化。【方法】通过16SrRNA基因对产卡拉胶酶的南极菌株进行种属鉴定,采用响应面法优化南极菌株产酶发酵条件。【结果】该南极菌属于交替单胞菌属(Alteromonas),命名为交替单胞菌R11-5。发酵条件优化结果显示,7个环境因子影响交替单胞菌R11-5的产酶量。利用Design-Expert软件中的Plackett-Burman设计实验,筛选出影响交替单胞菌R11-5产酶量的4个主要因素分别为培养温度、牛肉膏浓度、卡拉胶浓度和Ca~(2+)浓度。通过Box-Behnken设计和响应面分析得到交替单胞菌R11-5最佳产酶发酵条件为:温度15.0°C,牛肉膏浓度11.0 g/L,卡拉胶浓度3.0 g/L,Ca~(2+)浓度5.0 mmol/L。优化后发酵上清液酶产量达到87.193 U/mL,与优化前相比提高了1.8倍。【结论】响应面法提高了南极交替单胞菌R11-5卡拉胶酶的产量,为其开发应用提供了科学依据。     

低温脂肪酶产生菌的筛选、产酶发酵及粗酶性质研究

利用含有Tween 80的琼脂平板和摇瓶发酵法,从若尔盖高原土壤中筛选产脂肪酶菌株.通过菌落形态和菌体特征观察初步对菌种进行鉴定,得到一株产低温脂肪酶的适冷菌Pseudomonassp.DL-B,并设计正交试验对该菌株的产酶发酵培养条件进行了优化.摇瓶实验表明,该菌株最适产酶发酵培养基为:蔗糖10 g/L,蛋白胨20 ...     

Cloning and Expression of <Emphasis Type="Italic">lipP</Emphasis>, A Gene Encoding a Cold-Adapted Lipase from <Emphasis Type="Italic">Moritella</Emphasis> sp.2-5-10-1

A gene (lipP, 837 bp in length) coding for a cold-adapted lipase of psychrophilic bacterium Moritella sp. 2-5-10-1 isolated from Antarctic region was cloned and sequenced in this study. The deduced amino acid sequence revealed a protein of 278 amino acid residues with a molecular mass of 30,521. The primary structure of the lipase deduced from the nucleotide sequence showed consensus pentapeptide containing the active serine [Gly-Trp-Ser-Leu-Gly] and a conserved His-Gly dipeptide in the N-terminal part of the enzyme. These sequences were involved in the lipase active site conformation. Structure factors that would allow proper enzyme flexibility at low temperatures were discussed. It was suggested that the changes in the primary structure of the psychrophilic lipases compared to the thermophilic ones could account for their ability to catalyze lipolysis at temperatures close to 0°C. For expression, the sequence corresponding to the cold-adapted lipase of strain 2-5-10-1 was subcloned into the pET-28a expression vector to construct a recombinant lipase protein. Expression of the lipase by Escherichia coli BL21 (DE3) cells was observed as clear halos on 1% (vol/vol) tributyrin upon induction with IPTG at 25°C.     

Low-Temperature Lipase from Psychrotrophic Pseudomonas sp. Strain KB700A

We have previously reported that a psychrotrophic bacterium, Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at −5°C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca²⁺. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca²⁺-dependent lipase. Addition of Mn²⁺ and Sr²⁺ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca²⁺. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C₁₀ acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35°C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.     

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。     

Characterization of an alkaline lipase from Proteus vulgaris K80 and the DNA sequence of the encoding gene

Abstract A facultatively anaerobic bacterium producing an extracellular alkaline lipase was isolated from the soil collected near a sewage disposal plant in Korea and identified to be a strain of Proteus vulgaris . The molecular mass of the purified lipase K80 was estimated to be 31 kDa by SDS-PAGE. It was found to be an alkaline enzyme having maximum hydrolytid activity at pH 10, while fairly stable in a wide pH range from 5 to 11. The gene for lipase K80 was cloned in Escherichia coli . Sequence analysis showed an open reading frame of 861 bp coding for a polypeptide of 287 amino acid residues. The deduced amino acid sequence of the lipase gene had 46.3% identity to the lipase from Pseudomonas fragi .     

Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.     

酯酶产生菌的分离与酶学性质研究

从餐馆附近下水道收集到的土壤中分离获得6株酯酶产生菌,其中S2菌株活性最高,从其形态特征、生理生化试验以及16S rRNA序列分析等方面,初步鉴定S2菌株为假单胞属(Pseudomonas sp.)。对该菌株的部分酶学性质进行了研究,发现该菌株所产的酯酶最适反应温度为40℃,最适反应pH为8.0;且该酯酶在温度为60℃以下和pH 7.0～10.0具有良好的稳定性。