适冷微生物及其适冷机制研究进展

地球上许多生境为永久低温或季节性低温环境,适冷微生物在自然界中广泛存在。适冷微生物在环境净化、饲料、食品、奶制品、化妆品、皮革加工、洗涤等行业中具有广泛的应用前景。对适冷微生物的多样性、适冷的分子基础和适冷代谢机制进行了综述。     

低温微生物的冷适应机理及其应用

低温微生物广泛分布于极地、冰川、永久冻土和深海等寒冷环境,其冷适应能力是多种机理共同作用的结果,包括酶的低温催化活性、低温下膜流动性的保持、冷休克蛋白、抗冻蛋白以及抗冻保护剂等.低温微生物主要应用于催化低温发酵、表达热不稳定蛋白质、生产抗冻保护剂和冬季治理污水等领域.     

极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶发酵条件的优化

对极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶的发酵条件进行优化。结果表明:菌株QI-1的最适生长和产酶温度均为5℃;最佳接种量为1%;发酵培养基的最适初始pH和最佳装样量分别为5和10%;盐度为2%时对菌株的生长和产酶最为有利;麸皮和醋酸钠分别为最佳N源和C源;添加0.75%酪蛋白时菌株QI-1胞外蛋白酶的活性最高;10 mmol/L Mg2+和0.5%Tween-80有利于产酶。正交试验结果表明:菌株Pseudoalteromonassp. QI-1产蛋白酶较佳培养基配方(g/L)为麸皮5,酵母粉2.5,酪蛋白3,MgCl2.6H2O 3,KCl 1.5;发酵液比酶活为166.20 U/mL,较优化前提高了约56%。     

中国冰川1号产适冷蛋白酶耐冷菌的分离鉴定及产酶条件

从中国冰川 1号样品分离获得一株产适冷蛋白酶耐冷菌株SYP- A2 - 3,鉴定为蜡状芽孢杆菌 (Bacilluscereus)。该菌生长温度范围为 0～ 38℃ ,最适生长温度 2 5℃ ,而最适产酶温度为 15℃。所产蛋白酶为中性金属蛋白酶 ,最适催化温度为 4 2℃ ,低温催化活力较高 ,适宜作用pH为 7. 0～ 8 .5 ,SDS PAGE测定的分子量为 34 2kD。SYP A2 3产酶条件的研究结果显示酪蛋白是较好的氮源 ,葡萄糖、淀粉是较好的碳源 ,产酶最佳pH为 6. 5～ 7. 0 ,在优化的条件下 ,15℃摇瓶产酶达到 380 0U mL ,5L发酵罐通气培养产酶达 4 80 0U mL。     

产低温碱性蛋白酶海洋适冷菌SY的筛选

从连云港海域、港口、远洋捕捞船及鱼市等地采集的海水和各类海鱼、贝类等样品中分离到217株产蛋白酶的细菌,并从中得到1株产低温碱性蛋白酶的海洋适冷细菌—SY。研究表明,该菌株最适生长温度和最适产酶温度均在15℃左右,0℃下仍可生长;具有一定的耐盐性和嗜盐性,无盐条件不能生长,在3%的NaCl盐浓度时,生长达到最高峰;最适生长和最适产酶pH均为8.0;SY菌所产的蛋白酶可能为一种丝氨酸蛋白酶,酶最适作用温度为50℃,最适作用pH为9.0;酶的热稳定性差,50℃保温20min,酶活下降40%。     

低温细菌与古菌的生物多样性及其冷适应机制

低温细菌与古菌广泛分布于地球的低温环境,包括南极、北极及高山地带的冻土、低温土壤和荒漠、冰川、湖泊、海冰,以及深海、冰洞和大气平流层等.栖息在这些低温环境中的细菌与古菌具有丰富的多样性,主要为α,p和γ-Proteobacteria分支、CFB类群分支和革兰氏阳性细菌分支等.由于低温环境中的微生物流动性低,因而是研究微生物地理学理想的生态系统,有助于理解地球微生物的多样性、分布规律乃至形成机制.由于长期生活在冰冻环境中,低温细菌与古菌形成了多种适应低温环境的生理机制,如它们通过细胞膜脂类的组成来调节膜的流动性以维持正常的细胞生理功能;利用相容性溶质、抗冻蛋白、冰核蛋白及抗冰核形成蛋白等实现低温保护作用;产生冷激蛋白、冷适应蛋白和DEAD-box RNA解旋酶保持低温下RNA的正确折叠、蛋白质翻译等重要的生命活动;另外还产生低温酶,提高能量产生和储存效率等以适应低温环境.随着DNA序列分析技术的飞速发展,各类组学方法也用于揭示微生物全局性的冷适应机制.     

深海细菌及其适压机制

深海是以高压为主要特征的极端环境。在深海中适压生活的细菌在分类上多属于蛋白细菌(Pro-teobacteria)类群的γ分支。深海细菌对高压的适应可表现为嗜压或耐压,其适压的机制包括细胞膜脂成分中不饱和脂肪酸的增加,此外,细菌细胞的呼吸链系统也与耐压有关。     

南极地区海底沉积物中低温菌的鉴定与生长温度特征

从南极半岛及舍得兰群岛海底沉积物的13个样品中分离到23株细菌,进行了0、5、10、15、18 20、25℃温度梯度培养试验及耐盐度试验。由生长温度曲线得知,其中15株菌在0℃生长,依据菌株不同,其最适生长温度范围均在5—20℃以内;25℃下生长明显下降(9株)或缓慢下降(6株),属于耐冷菌。经鉴定,假单胞菌11株;弧菌1株;芽孢杆菌2株及黄杆菌1株。这15株菌中2,11(均为假单胞菌)及15(黄杆菌)的最适生长温度均为5℃;且前两株菌对中温较敏感;10 (假单胞菌);14(芽孢杆菌)的最适生长温度为10℃。本文还讨论了菌的分布与地区,以及与样点水源和距陆地远近间的相关性。     

北极海泥菌群的分离鉴定及生物学特性的初步研究

对从北极冰川海面下1500-4000m处的海泥样品中分离的8株冷适应细菌进行了生理特征和分子生物学研究。其中5株嗜冷菌、3株耐冷菌,利用16SrDNA通用引物对5株嗜冷菌基因组DNA进行扩增,测序得到其部分16SrDNA序列。经Blast调出与菌株16SrDNA同源的序列,按照Neighbor-Joining方法构建16SrDNA系统发育树。对8株细菌进行酶检测试验,结果表明其中有部分细菌产低温酶:N014产淀粉酶,R151产明胶酶,P371产纤维素酶。研究结果为进一步开发利用冷适应微生物产物提供参考依据。     

适冷纤维素降解微生物研究进展

该文概括了低温纤维素酶的特性,总结低温纤维素酶高产菌筛选、低温纤维素酶基因克隆、低温降解纤维素复合菌系选育的国内外研究进展,对今后纤维素酶高产菌的选育的发展趋势作了分析和综述.     

脂肪酸对人肺腺癌细胞膜流动性的影响

脂肪酸是细胞膜正常流动性的主要调节因素之一。本文报导了二种不同转移表型人肺腺细胞与九种不同脂肪酸共孵育后,对其细胞膜流动性的影响。结果表明,不同转移一夫肺腺癌细胞对各种脂肪酸有不同的敏感性,高转移癌细胞Ａｎｉｐ对棕榈酸和花生酸较敏感,而低转移癌细胞ＡＧＺＹ对棕榈烯酸和亚油酸较敏感。     

Inhibition of Topoisomerases by Fatty Acids

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure-activity relationships and mechanism of action were studied. Saturated fatty acids (C_6:0 to C_22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C_16:1 to C_22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C_18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.     

Sheep Erythrocyte Membrane Binding and Transfer of Long-Chain Fatty Acids

We have studied binding and membrane transfer rates of unsaturated long-chain fatty acids in sheep red cells, as previously done for human red cells, in order to elucidate the transport mechanism. Observed differences must be assigned to the different composition of the membrane in the two species. Equal surface areas of the membranes of the two species have similar binding capacities and affinities for palmitic-, linoleic-, oleic- and arachidonic acid at 37°C. The competitive bindings of linoleic- and arachidonic acid as well as the distribution of bound arachidonic acid on the two sides of the membrane are not different in the two species. However, the rate constants for membrane transfer in sheep are less than half of those measured previously for human ghosts. This finding is confirmed by the exchange efflux kinetics of ghosts containing albumin-bound fatty acid. Studies of sheep ghost membranes with oleic-, arachidonic- and linoleic acid reveal a proportionality between the membrane transfer rate constants and the number of fatty acid double bonds, as found previously for human ghost membrane, and the effect of double bonds is in harmony with a large negative activation entropy for diffusion through the membrane. The established replacement of lecithin by sphingomyelin with a low unsaturation fatty acid index in sheep membranes probably causes a lower transversal lipid phase fluidity. Double bonds diminish the flexibility of hydrocarbon chains and thus the large negative activation entropy of diffusion across the membrane. The smaller transfer rate constants of the three unsaturated fatty acids in sheep membranes support the hypothesis that the transfer is diffusion in protein defined annular lipid domains and not carrier mediated. Received: 24 February 1999/Revised: 10 June 1999     

Rat Brain Phosphatidyl-N,N-Dimethylethanolamine Is Rich in Polyunsaturated Fatty Acids

Phosphatidyl-N,N-dimethylethanolamine (PDME), an intermediate in the formation of phosphatidylcholine (PC) by the sequential methylation of phosphatidylethanolamine (PE), was purified from rat brain and its fatty acid (FA) composition compared with those of brain PC and PE. The proportion of polyunsaturated fatty acids (PUFAs) in the PDME (29.8%) was similar to that of PE (27.7%) and much greater than in PC (2.8%). Like the PUFAs of PE, the major PUFAs found in PDME were arachidonic acid (20:4) and docosahexaenoic acid (22:6). An isotopic method was developed to quantify the PDME purified from brain; a tritiated methyl group from CH3I was transferred to the PDME in the presence of cyclohexylamine to form [3H]PC, and the radioactivity of the PC was then counted. The concentration of rat brain PDME obtained using this method (33.0 +/- 1.8 micrograms/g brain) was very similar to that obtained using quantitative GLC analysis of its FAs (36.9 +/- 1.8 micrograms/g). The FAs in the PE and PC of rat brain synaptosomes were also analyzed; too little PDME was present in synaptosomes to permit similar analysis. The percentage of unsaturated FAs insynaptosomal PE was even higher (43.4 vs. 27.7) than that in PE prepared from whole brain. Since synaptosomes have a very high activity of phosphatidyl-N-methyltransferase, the enzyme complex that methylates PE to form PC, this enzyme may serve, in nerve endings, to produce a particular pool of PC, rich in PUFAs, which may have a distinct physiological function.     

杨梅核脂肪酸及蜡质成分分析

采用GC-MS联用技术,对杨梅(Myrica rubra)核中的脂肪酸及蜡质成分进行了分析.结果表明,杨梅核中脂肪酸和蜡质的总含量为31.10%,其中含有8种脂肪酸和3种长链烷烃,主要为棕榈酸(C16:0)28.22%、亚油酸(C182)14.4%、顺式-油酸(C18:1)25.74%、芥酸(C22:1)15.68%、十七烷5.2%、十八烷5.87%和十九烷4.71%,是一种优质和价廉的脂肪酸来源.     

侧柏叶子与种子脂肪酸的GC-MS比较分析研究

目的:研究柏子仁与侧柏叶的脂肪酸组成.方法:用GC-MS方法对侧柏叶子与种子油进行定性鉴定和定量分析.结果:鉴定了柏子仁油中的8种脂肪酸,占脂溶性成分的93.56%;侧柏叶子油中12种脂肪酸,占脂溶性成分的93.39%.柏子仁饱和脂肪酸主要是十六烷酸(8.11%)、硬脂酸(6.08%);不饱和脂肪酸主要为亚油酸(24.59%)、亚麻酸(59.77%),占脂肪酸的83.14%.侧柏叶子饱和脂肪酸饱和脂肪酸主要为十六烷酸(14.70%)、乙酸(3.20%)、十七烷酸(2.50%);不饱和脂肪酸主要为二十二碳四烯酸(40.48%)、亚油酸(10.69%)、亚麻酸(17.62%).结论:柏子仁和侧柏叶均含有合理的脂肪酸组成.     

Isolation and Characterization of Two Fatty Acid Binding Proteins from Mouse Brain

Abstract: Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross-reacted with antisera to recombinant liver L-FABP. One protein, designated brain H-FABP, migrated on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H-FABP bound NBD-stearic acid and cis -parinaric acid with K _D values near 0.02 and 0.5 µ M , respectively. Brain H-FABP cross-reacted with affinity-purified antisera to recombinant heart H-FABP. The second protein, mouse brain B-FABP, migrated on tricine SDS-PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B-FABP bound NBD-stearic acid and cis -parinaric acid with K _D values near 0.01 and 0.7 µ M , respectively. The brain B-FABP doublet was immunoreactive with affinity-purified antibodies against recombinant mouse brain B-FABP, but not with affinity-purified antibodies against heart H-FABP. [³H]Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B-FABP and brain H-FABP represented 0.1 and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H-FABP and brain B-FABP.     

植物脂肪酸的生物合成与基因工程

植物脂肪酸既具重要生理功能,又有巨大食用和工业价值。其生物合成途径较为复杂,涉及乙酰－ＣｏＡ羟化酶、脂肪酸合成酶、脂肪酸去饱和酶和脂肪酸延长酶等一系列酶。近年来,对脂肪酸生物合成途径进行了大量研究,克隆出许多相关基因,初步阐明了脂肪酸合成规律,并在此基础上开展了利用基因工程技术调控脂肪酸合成研究,取得可喜进展。本文详细介绍了植物饱和脂肪酸、不饱和脂肪酸和超长链脂肪酸的生物合成与基因工程研究的新结果     

微生物合成奇数链脂肪酸研究进展*

奇数链脂肪酸(odd-chain fatty acids,OCFA)在自然界分布广泛但含量低,在食品、医药健康和工业等领域有着巨大的应用潜力。目前获取OCFA的方法主要为提取法和化学合成法,但成本高、效率低,而通过微生物发酵有望实现OCFA大规模工业生产。总结OCFA的应用范围和天然合成OCFA的微生物种类,详述微生物合成OCFA的代谢途径,并从基因工程策略和发酵调控策略两方面综述目前提升OCFA产量的研究现状,旨在为利用合成生物学策略改造和提升微生物合成OCFA的能力提供较为系统的理论依据。