食品中低温微生物的适冷机制研究进展

低温贮藏是延长食品货架期、维持食品鲜度和质量安全的重要方法,然而仍有部分微生物能适应低温环境,使食品发生腐败变质。主要从细胞膜、适冷酶、冷休克蛋白、冷适应蛋白、代谢水平及低温防护剂等角度阐述国内外食品中低温微生物适冷机制的研究进展,为低温微生物在食品领域的应用与防护提供参考。     

低温微生物及其酶类的研究概况

广泛分布在地球寒冷生境 ,如南北两极、高山、深海以及冰川中的低温微生物 ,不但为研究低温生态系统、生命起源与进化以及生物适冷机制提供了丰富的材料 ,同时在生物工程方面也具有潜在的巨大开发价值。国内外越来越多的科研人员对低温微生物及其产物的研究表现出了浓厚的兴趣。关于细胞膜和低温酶的研究 ,是目前微生物适冷机制研究中的 2个热点。就低温微生物的研究现状和适冷机制以及低温酶类的研究进行了综述。     

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。     

嗜冷酶及其工业应用

生物圈中超过 80 %的地方温度低于5℃ ,在生态学上低温环境的影响范围更广。生活在这些低温环境中的嗜冷菌对低温有特殊的适应性。相对于嗜温菌而言 ,嗜冷微生物在低温条件下调节其膜流动性和膜通透性的能力更强 ,含有更多的不饱和脂肪酸和短链脂肪酸 ,还可通过合成冷激蛋白来响应冷激作用。嗜冷菌也可通过基因调控和自身的抗生素作用来适应低温。近来有研究报道 ,南北极严寒地区的海洋鱼类依靠血液中的抗冻蛋白来适应低温。生物体内的大多数生化反应都是由酶催化而成的 ,所以考虑嗜冷微生物的适应机制时首先应研究嗜冷酶的适冷机制。1 .嗜…     

常温土壤中耐冷菌的分离、鉴定及产酶分析

目的:从常温土壤中筛选冷适应微生物,并进行初步鉴定和产低温酶分析。方法:采集吉首大学校园内土壤样品,通过低温富集培养筛选冷适应微生物;通过形态观察、生理生化特性检测和基于16S rRNA基因序列的系统发育分析,对分离的菌株进行初步鉴定;利用平板筛选法检测其产低温酶特性。结果:分离获得6株耐冷细菌JSBP-1～JSBP-6,初步鉴定其分属假单胞菌属(Pseudomonas)、紫色杆菌属(Janthinobacterium)和节杆菌属(Arthrobacter);在5℃和15℃培养条件下,菌株JSBP-1产蛋白酶能力较强,JSBP-2和JSBP-6产淀粉酶能力较强,JSBP-5仅在5℃条件下有较强的产脂肪酶特性。结论:常温土壤中存在一定数量的冷适应微生物,其中假单胞菌是其优势菌群之一。这类适冷微生物菌群具有潜在的生产低温酶能力。     

低温纤维素酶的研究进展

低温纤维素酶在低温下仍具有较高酶活力及较高催化效率,在应用中能够缩短处理工艺时间,节省加热或冷却费用,且易失活,有着中高温纤维素酶无法比拟的优势。现针对微生物低温纤维素酶的研究现状,包括菌种来源、分离鉴定、酶学性质、适冷结构及机理研究和基因工程等方面进行综述。     

适冷酶:性能与应用

由耐寒微生物产生的适冷酶显示出较高的催化活力,并常与热敏感性有关。运用X射线的结晶学理论,这一特性已开始逐渐为人所知,且控制它们对低温适应能力的机制各不相同。这些酶的应用为生物技术提供了巨大的发展潜力,例如在清洁剂和食品行业中用于生产精细化学品以及在生物环保处理中的应用。     

低温微生物在污水处理中的应用分析

低温微生物作为极端环境微生物研究的一大热点,自20世纪70年代以来,其研究应用就得到了越来越广泛的关注。低温微生物凭借一系列耐冷机制,可有效抵御低温对其细胞生存生长的胁迫,保障其在低温环境下仍能够生存繁殖。文章首先阐述了低温微生物的相关内涵及其适冷机制,然后探讨了低温微生物在污水处理中的实际应用,以期为低温微生物在实际生产生活中的应用提供参考。     

低温酶及其冷适应性机制研究进展

生活在极地、深海等常年低温环境中的低温微生物和部分变温生物,能够通过产生低温酶来适应在低温环境中的生长、繁殖和代谢。这些酶在低温或常温条件下具有很高的催化活性,对热却很敏感,在高温时能够快速失活,因此在实际的生产和生活中具有广泛的应用前景。基于这些特性,低温酶的冷适应性机制研究成为了当前的一个热点。较系统地综述了低温酶的来源、特点、生产状况以及具有代表性的几种低温酶的冷适应性机制。     

低温微生物适应低温的分子机制

根据Ｍｏｒｉｔａ和Ｒｕｓｓｅｌ的定义 ,低温微生物可分为两类 :一类是其最高生长温度低于2 0℃的微生物称嗜冷菌 (ｐｓｙｃｈｒｏｐｈｉｌｅｓ) ,另一类是指在 0～ 40℃可以生长的微生物称耐冷菌 (ｐｓｙｃｈｒｏｔｒｏｐｈｓ)。这些微生物体内的温度与环境温度很接近。尽管低温对生化反应有着强烈的负效应 ,但低温微生物在低温下却能成功地生长与繁殖 ,它们因此产生了各种各样的适应机制 ,主要是细胞膜、蛋白质、酶分子水平上发生了精细的组成和结构的变化[1] ,因此弥补了低温对生长的有害影响。催化生物体内所有生化反应的酶是生物体适…     

Cellulase recycling in biorefineries—is it possible?

On a near future, bio-based economy will assume a key role in our lives. Lignocellulosic materials (e.g., agroforestry residues, industrial/solid wastes) represent a cheaper and environmentally friendly option to fossil fuels. Indeed, following suitable processing, they can be metabolized by different microorganisms to produce a wide range of compounds currently obtained by chemical synthesis. However, due to the recalcitrant nature of these materials, they cannot be directly used by microorganisms, the conversion of polysaccharides into simpler sugars being thus required. This conversion, which is usually undertaken enzymatically, represents a significant part on the final cost of the process. This fact has driven intense efforts on the reduction of the enzyme cost following different strategies. Here, we describe the fundamentals of the enzyme recycling technology, more specifically, cellulase recycling. We focus on the main strategies available for the recovery of both the liquid- and solid-bound enzyme fractions and discuss the relevant operational parameters (e.g., composition, temperature, additives, and pH). Although the efforts from the industry and enzyme suppliers are primarily oriented toward the development of enzyme cocktails able to quickly and effectively process biomass, it seems clear by now that enzyme recycling is technically possible.     

Cold adaptation of microorganisms

Psychrophilic and psychrotrophic microorganisms are important in global ecology as a large proportion of our planet is cold (below 5 degrees C); they are responsible for the spoilage of chilled food and they also have potential uses in low-temperature biotechnological processes. Psychrophiles and psychrotrophs are both capable of growing at or close to zero, but the optimum and upper temperature limits for growth are lower for psychrophiles compared with psychrotrophs. Psychrophiles are more often isolated from permanently cold habitats, whereas psychrotrophs tend to dominate those environments that undergo thermal fluctuations. The molecular basis of psychrophily is reviewed in terms of biochemical mechanisms. The lower growth temperature limit is fixed by the freezing properties of dilute aqueous solutions inside and outside the cell. In contrast, the ability of psychrophiles and psychrotrophs to grow at low, but not moderate, temperatures depends on adaptive changes in cellular proteins and lipids. Changes in proteins are genotypic, and are related to the properties of enzymes and translation systems, whereas changes in lipids are genotypic or phenotypic and are important in regulating membrane fluidity and permeability. The ability to adapt their solute uptake systems through membrane lipid modulation may distinguish psychrophiles from psychrotrophs. The upper growth temperature limit can result from the inactivation of a single enzyme type or system, including protein synthesis or energy generation.     

Moritella cold-active dihydrofolate reductase: are there natural limits to optimization of catalytic efficiency at low temperature?

Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2 degrees C or less. The enzyme is monomeric (Mr=18,291), 55% identical to its Escherichia coli counterpart, and displays Tm and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0 degrees C. At mesophilic temperatures the apparent Km value for dihydrofolate is 50- to 80-fold higher than for E. coli, Lactobacillus casei, and T. maritima dihydrofolate reductases, whereas the apparent Km value for NADPH, though higher, remains in the same order of magnitude. At 5 degrees C these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency (kcat/Km) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.     

中温（37℃）纤维素分解菌的筛选及混合培养研究

目的:筛选纤维素分解菌,构建复合微生物菌系进行混合培养,获得降解纤维素能力强的复合菌系.方法:从高温阶段堆肥样品、腐熟肥料、牛粪和土壤中筛选出能较好降解纤维素的菌株,进行单独与混合发酵培养,测其酶活.结果:获得降解纤维素能力较强的细菌3株、霉菌4株、放线菌2株.按不同的接种比例构建了4组复合微生物菌系,4个组合的滤纸失重率分别达到41.52%、44.94%、41.82%、37.11%,液体发酵的平均CMC酶活分别为624U/g、988U/g、769U/g、1041U/g,固体发酵的平均CMC酶活分别为4240U/g、5289U/g、4807U/g、5344U/g.结论:综合分析复合菌系滤纸条降解能力、CMC酶活、FAP酶活表明,组合二分解纤维素能力明显强于其他几个组合.     

North Western Spain hot springs are a source of lipolytic enzyme-producing thermophilic microorganisms

Several hot springs in Galicia (North Western Spain) have been investigated as potential sources of lipolytic enzyme-producing thermophilic microorganisms. After isolating 12 esterase producing strains, 9 of them were assured to be true lipase producers, and consequently grown in submerged cultures, obtaining high extracellular activities by two of them. Furthermore, a preliminary partial characterization of the crude lipase, obtained by ultrafiltration of the cell-free culture supernatant, was carried out at several pH and temperature values. It is outstanding that several enzymes turned out to be multiextremozymes, since they had their optimum temperature and pH at typical values from thermoalkalophiles. The thermal stability in aqueous solution of the crude enzymes was also assayed, and the influence of some potential enzyme stabilizing compounds was tested. Finally, the viability of the selected microorganisms has been demonstrated at bioreactor scale.     

Molecular characterization of inulosucrase from Leuconostoc citreum: a fructosyltransferase within a glucosyltransferase

The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme. Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase. Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E. coli and retained their ability to synthesize inulin but lost thermal stability. This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.     

Genome Sequence of a Cold-Adaptable Sulfamethoxazole-Degrading Bacterium,Pseudomonas psychrophila HA-4

Pseudomonas psychrophila HA-4 is a cold-adaptable, sulfamethoxazole-degrading bacterium. The genes related to its cold adaptation mechanism and sulfamethoxazole metabolism were unknown. We present the draft genome of strain HA-4. It could provide further insight into the sulfamethoxazole-degrading mechanism of strain HA-4.     

低温秸秆降解复合微生物菌剂的研究进展

我国东北地区冬季寒冷,秸秆产量巨大,但综合利用率较低,利用高酶活性微生物将低温环境中的秸秆降解变废为宝,是一项循环利用的有效途径。研究表明,通过生物学技术手段,筛选高酶活性菌株,深入研究降解机理,优化功能微生物培养条件,提高纤维素酶活性,是提高降解率,秸秆资源化利用的最佳途径。复合微生物菌剂产生的酶活值普遍高于单一微生物菌,真菌菌丝体产生的酶活值高于细菌。实际应用中,选择适合的复合菌剂是低温环境下提高秸秆降解效率的有效途径。系统地归纳了低温条件下降解秸秆的微生物技术、分析了不同条件下降解秸秆的菌株类型和促进秸秆纤维素降解菌酶活力特征、并总结了低温环境下生物菌剂降解秸秆的技术应用效果,旨为低温环境下秸秆的资源化利用提供一定的技术参考。     

细菌苏氨酸脱水酶受L-异亮氨酸反馈抑制的关键在于其调控结构域

苏氨酸脱水酶是细菌L-异亮氨酸合成途径中的关键酶,酶活力受终产物L-异亮氨酸的反馈抑制。苏氨酸脱水酶包含催化结构域及调控结构域,其中调控结构域所起的作用有待进一步研究。本文在大肠杆菌中分别克隆表达了四种不同的苏氨酸脱水酶:来自谷氨酸棒杆菌的苏氨酸脱水酶CgIlvA及其不合调控结构域的突变体CgIlvA~M和来自大肠杆菌的苏氨酸脱水酶EcIlvA及其不合调控结构域的突变体EcIlvA~M。通过蛋白纯化和酶活分析发现,CgIlvA~M和EcIlvA~M的酶活力比CgIlvA和EcIlvA的酶活力有所降低,但它们不再受L-异亮氨酸的反馈抑制,说明L-异亮氨酸对苏氨酸脱水酶的反馈抑制是通过其调控结构域来实现的。