微生物脂肪酶生物学特性及分离纯化研究进展

微生物脂肪酶具有化学结构选择性、区域选择性及对映性,在有水和无水条件下均可高效催化反应,为了在工业中应用微生物脂肪酶,对微生物脂肪酶的氨基酸序列、二级结构和三级结构等生物学结构,微生物脂肪酶的与底物结合、酶-酰基共价中间体复合物的形成和弦基脱除等催化机制,以及微生物脂肪酶的双水相萃取分离法、膜分离法、硫酸铵-丙酮协同沉淀法、免疫纯化技术和界面亲和层析技术等分离纯化技术等方面进行了综述,并根据当前国内外研究现状对微生物脂肪酶发展进行了展望并提出几点发展建议。     

各类微生物脂肪酶酶学性质及应用研究进展

微生物脂肪酶作为一类功能强大的催化剂,越来越受到人们的关注。微生物脂肪酶种类繁多,广泛存在于霉菌、酵母和细菌中,且不同微生物来源的脂肪酶其酶学性质、基因结构各不相同。对目前国内外研究较多的根霉脂肪酶、曲霉脂肪酶、青霉脂肪酶、毛霉脂肪酶、地霉脂肪酶、酵母脂肪酶和细菌脂肪酶的最适作用温度、最适pH及pH稳定性、底物特异性、酶活性影响、基因结构及其应用等方面进行了综述，并论述了食品加工业中脂肪酶的研究进展。     

铜绿假单胞菌脂肪酶基因在枯草芽孢杆菌中的克隆与表达

利用PCR方法从分泌脂肪酶的铜绿假单胞菌基因组DNA中扩增得到了脂肪酶基因,并测定其核苷酸序列,利用基因重组技术构建了脂肪酶基因的分泌表达载体,并在枯草芽孢杆菌中进行了分泌表达。SDS聚丙烯酰胺凝胶电泳表明,表达蛋白占发酵液中蛋白的25%,采用NaOH碱滴定法测定其发酵液酶活为15.26U/mL。     

微生物脂肪酶及其催化合成芳香酯研究进展

微生物脂肪酶可以催化酯类化合物的分解、合成和酯交换，因而被广泛应用于食品、精细化工和医药等工业中，近年来，随着界面酶学、有机相催化以及固化技术的不断深入和发展，微生物脂肪酶的诸多新特性正在被发现，其应用领域也在不断拓展。国内外的研究结果表明，用微生物脂肪酶可以催化合成多种芳香酯，并且在有机相中微生物脂肪酶的催化活性和稳定性较水溶液中明显提高；用微生物脂肪酶催化合成芳香酯可以克服用化学方法合成带来的问题，是一种芳香酯合成的有效方法。     

微生物脂肪酶资源挖掘研究进展

脂肪酶广泛分布于动物、植物和微生物中,工业化脂肪酶主要来源于微生物。脂肪酶制剂的进一步工业化 应用受限于生产成本和脂肪酶的温度和pH值耐受性、活性、专一性和溶剂耐受性等酶学性质,虽然微生物脂肪酶 基因和蛋白相关资源信息已经非常丰富,但适合食品、药品和能源等工业应用的脂肪酶制剂品种依然较少,研究者 仍然在为新型和理想的脂肪酶制剂资源的挖掘而努力。本文重点综述了微生物脂肪酶资源挖掘的主要研究方法,主 要包括通过环境筛选和宏基因组筛选挖掘新的具有优良特性的脂肪酶;脂肪酶微生物生产菌株的改良、脂肪酶基因 在重组工程菌株中的重组表达和优化、蛋白质工程方法对脂肪酶蛋白进行改造、脂肪酶固定化和催化工艺的改良 等。     

各类微生物脂肪酶酶学性质及应用的研究进展

微生物脂肪酶作为一类功能强大的催化剂,越来越受到人们的关注。微生物脂肪酶种类繁多,广泛存在于霉菌、酵母和细菌中,且不同微生物来源的脂肪酶其酶学性质、基因结构各不相同。对目前国内外研究较多的根霉脂肪酶、曲霉脂肪酶、青霉脂肪酶、毛霉脂肪酶、地霉脂肪酶、酵母脂肪酶和细菌脂肪酶的最适作用温度、最适pH及pH稳定性、底物特异性、酶活性影响、基因结构及其应用等方面进行了综述,并论述了食品加工业中脂肪酶的研究进展。     

以分子微生物学技术提升传统绍兴酒的酿造工艺

提出了制约传统绍兴酒酿造技术水平提高的四个主要问题；阐述了传统绍兴酒酿造过程中的微生物群系的变化和质量风格影响因素十分复杂；提出了以微生物的DNA核苷酸序列、基因组等为研究对象的分子微生物学可以大规模地平行处理多种微生物的种群动态变化规律，以进一步提升传统绍兴酒的酿造工艺。     

脂肪酶的研究进展

随着生物技术的日新月异，酶固定化技术，界面酶学和非水酶学研究与应用的突破性进展，使得人们对微生物脂肪酶产生了极大的兴趣。论述脂肪酶的种类和高产菌珠以及脂肪酶的一些性质和应用。     

用于油脂改性的1,3—定向脂肪酶

对微生物１，３－定向脂肪酶催化的油脂定向酯交换的性质、应用及１，３－定向脂肪酶产生菌的选育作了较详尽的综述，简要介绍了当前脂肪酶的纯化和固定化技术，酶生物技术的引入将为油脂工业展现广阔的前景。     

用于油脂改性的1，3－定向脂肪酶

对微生物１，３－定向脂肪酶催化油脂定向酯交换的性质、应用及１，３－定向脂肪酶产生菌的选育作了较详尽的综述，简要介绍了当前脂肪酶的纯化和固定化技术，酶生物技术的引入将为油脂工业展现广阔的前景。     

Identification of a triacylglycerol lipase gene family in Candida deformans: molecular cloning and functional expression

The yeast Candida deformans CBS 2071 produces an extracellular lipase which was shown to catalyse the production of various esters by the esterification of free fatty acids, even in the presence of a large molar excess of water. To clone the gene encoding this extracellular lipase, Saccharomyces cerevisiae was transformed with C. deformans genomic libraries and screened for lipolytic activity on a medium containing rapeseed oil emulsion and rhodamine B. Three members of a lipase gene family (CdLIP1, CdLIP2 and CdLIP3) were cloned and characterized. Each deduced lipase sequence has a Gly-His-Ser-Leu-Gly-(Gly/Ala)-Ala conserved motif, eight cysteine residues and encodes an N-terminal signal sequence. MALDI-TOF mass spectrometry analysis of a proteolytic digest of the lipase produced was used to obtain experimental evidence that the CdLIP1 gene encoded the extracellular lipase. Recombinant expression studies confirmed that the cloned genes encoded functional lipases. The three lipases are very similar to lipases from the related species Yarrowia lipolytica. Significant homologies were also found with several yeast and fungal lipases. As C. deformans CBS 2071 was previously considered to be synonymous with Y. lipolytica, the strains were compared for the extent of nucleotide divergence in the variable regions (D1/D2) at the 5'-end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region has diverged sufficiently to suggest that C. deformans is a separate species. The nucleotide sequences of the CdLIP1, CdLIP2 and CdLIP3 genes will appear in the EMBL nucleotide sequence database under Accession Nos AJ428393, AJ428394 and AJ428395, respectively.     

An extracellular lipase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ALIP1 gene and characterization of the purified recombinant enzyme

The lipase-encoding Arxula adeninivorans ALIP1 gene was isolated using fragments of lipase isolates obtained by trypsin digestion for the definition of oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1347 bp encoding a 420 amino acid protein of some 50 kDa preceded by an N-terminal 28 prepro-secretion sequence. The deduced amino acid sequence was found to be similar to the lipases from Candida albicans and C. parapsilosis (34-38% identity) and more distantly related to other lipases. The sequence contains the consensus pentapeptide motif (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases. The expression of the gene is regulated by carbon source. In media supplemented with Tween 20, induction of the ALIP1 gene and accumulation of the encoded lipase in the medium is observed, thus demonstrating gene regulation by lipophilic compounds. The enzyme characteristics are analysed from isolates of native strains as well as from those of recombinant strains expressing the ALIP1 gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins a molecular mass of 100 kDa was determined, indicating a dimeric structure, a pH optimum at pH 7.5 and a temperature optimum at 30 degrees C. The enzyme hydrolyses all ester bonds in all triglyceride substrates tested. Middle-sized chain fatty acids are more efficiently hydrolysed than short- and long-chain fatty acids, with the highest activity on C8/C10 fatty acid esters pNP-caprylate, pNP-caprate and tricaprylin.     

脂肪酶在纺织工业中的应用

从脂肪酶的特点及其应用范围出发,对目前脂肪酶在纺织工业中的应用研究进行了综述.主要介绍了脂肪酶在羊毛纤维毡缩性的改善、丝绸纤维上的油脂和蜡质的去除、棉纤维的脱浆以及涤纶纤维性能的改善等方面的作用及研究进展.探讨了脂肪酶在纺织工业应用研究中存在的几个问题,并对其在纺织工业中的进一步应用做了展望.     

施氏假单胞菌中不同细胞组分的脂肪酶合成活性研究

本研究以多穗柯为材料,采用同时蒸馏萃取法（SDE）提取挥发性组分,通过气质联用仪（GC-MS）分析挥发性组分组成,并以Hepa1c1c7小鼠肝癌细胞模型,以细胞存活率大于50%,诱导醌还原酶（QR）活性等于或大于2为活性指标,研究多穗柯挥发性组分及其主要单体物质的抗癌活性。结果显示,GC-MS共鉴定出80种多穗柯挥发性物质,占总挥发性组分相对百分含量的92.56%,其中酮类、醇类和醛类物质是多穗柯挥发性组分的主要类型,其主要成分有香叶基丙酮、β-紫罗酮、壬醛和桉油烯醇等。抗癌活性鉴定表明,多穗柯挥发性组分的浓度为(27.50±0.10) μg/mL时可诱导QR活性达到倍增,具有较强的抗癌活性;其中芳樟醇、香叶基丙酮和壬醛三个主要挥发性单体成分诱导QR活性达到倍增的浓度分别为(54.53±0.10) μg/mL、(283.10±0.10) μg/mL和(297.77±0.12) μg/mL。     

Effect of hydrocarbon-water interfaces on synthetic and hydrolytic activities of lipases

We have developed a new method of lipase activation for interesterification, in which lipase, in contact with a hydrocarbon-water interface, has been found to have high interesterification activity in an anhydrous solvent. We have applied this activation method to various lipase and obtained high synthetic activity in n-hexane, and have investigated the effect of various hydrocarbon-water interfaces on the synthetic and hydrolytic activities of lipases. The esterification and/or interesterification activity of lipases tested was improved by this activation method, using an n-tetradecane-water interface. From the initial group of lipases, three representative lipases (from Rhizopus japonicus, Chromobacterium viscosum and porcine pancreas) were selected for further study. The effect of various hydrocarbon-water interfaces on synthetic (interesterification or esterification) activity was studied. We demonstrated that the resulting synthetic activity was affected by the choice of hydrocarbon-water interface and that there were differences in the effects of interfaces on the synthetic activity of these lipases.     

脂肪酶位置选择性的影响因素

为开发高效、绿色、低成本的脂肪酶应用于结构脂质的制备，从内源性(脂肪酶的来源、基因序列、空间位阻)和外源性(底物空间结构、反应介质、固定化条件及反应pH)两方面讨论了脂肪酶位置选择性的影响因素。脂肪酶催化特异性由内源性因素和外源性因素共同决定。改变底物空间结构、反应介质、固定化条件和反应pH,常被用作提高脂肪酶催化性能的处理方式。只有充分考虑内外两方面因素的影响，才能充分提高脂肪酶的位置选择性和催化效率。该综述有望为高选择性脂肪酶的开发开辟新的研究方向，为后续结构脂质的高效合成提供启发性的研究思路。     

Statistical Patterns of Lipase Activities on the Release of Short-Chain Fatty Acids in Cheddar Cheese Slurries

Twenty-five commercial food grade and analytical grade lipases were used to study the patterns of release of short-chain fatty acids (FFA) from milk fat in cheese slurries. Principal Component Analysis showed that there were four distinctive groups indicated by the FFA ratios and five groups indicated by the FFA concentrations. Average Linkage Cluster Analysis also showed similar results although the patterns of FFA released were a matter of distances defined statistically between groups of lipases. All the lipases tested had distinctive specificities in hydrolyzing short-chain FFA from milk fat, and their specificities on the FFA release were reflected on the groupings.     

不同来源脂肪酶的催化反应性质比较

研究了三种脂肪酶的催化反应性质,包括水解作用pH、温度适宜范围,酶的酸碱稳定性和热稳定性,酶催化水解作用的底物脂肪酸特异性和酯键位置特异性,及对逆向反应的催化作用等。对三种酶的催化反应的机制进行了比较和讨论。     

超临界体系酶催化制备甘油二酯及其纯化

以二氧化碳为流体,在超临界体系中用脂肪酶催化大豆油脂与甘油反应制备甘油二酯及其纯化研究。选取Lipozyme RMIM、Novozyme 435、Lipozyme TLIM 三种固定化脂肪酶为试验酶进行酯化反应,通过单因素试验,分别确定3种酶的最佳工艺条件,3种酶的最佳添加量分别为2.5%、3%、8%;反应温度分别为65、70、65℃,反应时间分别为7、8、9h,底物比均为2:1,得甘油二酯含量分别为68.6%、67.7%、64.8%。采用二级分子蒸馏工艺对超临界体系生产的甘油二酯混合物进行纯化,甘油二酯产品纯度从68.6%提高到90.4%,产品得率为60.0%。综合考虑,在工业生产中,建议用脂肪酶Lipozyme RMIM。