米曲霉产胞外脂肪酶培养条件的优化

对米曲霉产胞外脂肪酶的培养条件进行了优化。筛选出麦芽糖为最佳碳源,蛋白胨为最佳氮源,平平加O为最佳的表面活性剂。该菌株产脂肪酶的最适培养条件为:麦芽糖0 5 % ,蛋白胨0 5 % ,橄榄油1 0 % ,平平加O 0 0 32 % ,发酵液初始pH值为6。     

内生黑曲霉产脂肪酶条件研究及其粗酶酶学特性

针对新疆酿酒葡萄中获得的1株产脂肪酶的内生黑曲霉菌株C2J6,研究了该菌株的产酶条件及酶学特性。结果表明,该菌适宜的产酶条件为1%的乳糖为碳源,1%的蛋白胨为氮源,培养基初始pH值为8.0,培养温度35℃,培养时间约72h,此时所产脂肪酶的活力可达18.75U/mL。该菌所产脂肪酶粗酶液的最适反应温度为40℃,最适反应pH值为7.0,为中温中性酶;在50℃保温1h酶活力保留54.55%,具有良好的热稳定性;在pH值3.0~7.0范围内较稳定,有一定的耐酸性。金属离子Mn2+对酶活力有促进作用,Zn2+、Fe2+、Cu2+对酶活力有抑制作用,K+、Ca2+、Na+、Mg2+对酶活力影响不大。该酶在以葵花油和谷物调和油为底物时,酶活分别为228%和180%,明显高于橄榄油和油烟机废油。     

黑曲霉脂肪酶合成单月桂酸甘油酯

从黑曲霉中提出了一种具有很高催化活性和选择性的脂肪酶,为证明这种脂肪酶的高选择性,用此酶直接催化甘油和月桂酸反应合成单月桂酸甘油酯,并且优化了反应的工艺参数。实验表明,采用甘油月桂酸摩尔比为1∶1.5,脂肪酶与底物质量比为0.5%,水与底物质量比为3%的条件在50℃下反应12h,可使月桂酸转化率达到91.2%,单酯含量高达70%。     

疏棉状嗜热丝孢菌耐热脂肪酶在无孢黑曲霉中的高效表达

综合考虑密码子偏爱性、RNA稳定性以及自由能等因素,对疏棉状嗜热丝孢菌(Thermomyces lanuginosus)耐热脂肪酶(tll)基因序列进行密码子优化并全基因合成。将合成的tll基因连接到克隆载体,然后亚克隆到表达载体pHGWPT-tll,采用根癌农杆菌介导转化无孢黑曲霉SH-2。经过三丁酸甘油酯平板、SDS-PAGE以及Western Blotting鉴定,成功表达了tll基因。发酵72h,转化子脂肪酶酶活最高达36U/mL。转化子经等离子诱变得到的突变株的脂肪酶比活力比出发菌株提高约37%。     

黑曲霉C2J6脂肪酶的保存稳定性研究

为提高黑曲霉（Aspergillus niger）C2J6脂肪酶的储存和后续使用的稳定性,通过单因素试验和Box-Behnken响应面优化试验, 考察了其在糖类、醇类、盐类稳定剂中的保存稳定性。 结果表明,添加单一稳定剂,相对酶活保持在63.50%左右;而复合稳定剂因协同效 应的存在更有利于酶的稳定化,酶液中添加丙三醇3.54 mol/L,硫酸镁0.14 mol/L,果糖5%,此条件下保存48 h后相对酶活能达到72.90%。 关键词：     

双有机溶剂中黑曲霉脂肪酶催化阿魏酸的酯化反应

在双有机溶剂体系中,用黑曲霉脂肪酶催化合成阿魏酸油醇酯,考察酶浓度、温度和底物摩尔比等因素对酯化反应的影响。结果表明:在异辛烷/丁酮体系中,当反应温度为60℃,阿魏酸和油醇的摩尔比为1∶8,即阿魏酸浓度为0.39 mg/mL和油醇浓度为4.3 mg/mL,脂肪酶浓度为0.2 g/mL时,转化率为97.6%;而在环己烷/丁酮体系中,当反应温度为60℃,阿魏酸和油醇的摩尔比为1∶8,即阿魏酸浓度为0.49 mg/mL和油醇浓度为5.37 mg/mL,脂肪酶浓度为0.25 g/mL时,转化率为91.0%。     

双氧水对米赫根毛霉脂肪酶在米曲霉中重组表达的调控研究

米赫根毛霉脂肪酶RML是具有广泛应用价值的重要微生物脂肪酶。本研究对双氧水调控米曲霉RML转化子ONL1表达脂肪酶进行了研究。荧光定量PCR和SDS-PAGE表明,在转化子培养过程中,维持培养体系中10 mmol/L双氧水2 h,使转化子ONL1的脂肪酶活力提高5倍。在双氧水处理过程中,RML的翻译未受影响,其表达水平的提高源于双氧水对其转录水平的调控。因此,双氧水调控melO启动子控制的外源基因的表达体现在转录水平。由于双氧水易挥发,导致其效果低于延长培养时间。为了在较短培养时间内获得高酶活,应在培养液中持续添加双氧水（10 mmol/L）。基于米曲霉转化子脂肪酶活力的分析和qPCR检测,确定以下策略可用于提高米曲霉中RML的表达水平：脂肪酶RML基因的密码子优化、信号肽序列优化、连续分批培养。     

培养条件对黑曲霉脂肪酶选择性合成1,3甘油二酯的影响

从前期筛菌工作中得到的一株能高选择性合成1,3-甘油二酯（1,3-DAG）的产胞内脂肪酶的黑曲霉GZUF36菌株出发,分别研究了培养时间、温度、接种量、装液量和转速对该菌摇瓶发酵的影响,得出合适的培养条件为培养时间72 h,接种量3%,装液量40 mL/250 mL,培养温度28 ℃,转速180 r/min,在此条件下1,3-DAG的得率和选择性分别为23.87%和82.96%。同时也比较了摇瓶发酵和5 L发酵罐发酵的异同,相比于摇瓶发酵,5 L发酵罐发酵的1,3-DAG的得率有所提高,但是发酵罐发酵的时间有所延长。     

Lipid Fractions of Dry Fermented Sausages Change when Starter Culture and/or Aspergillus Lipase are Added

Six levels of Aspergillus sp. lipase were evaluated and 0.400u/g was selected. A sausage with a starter culture (Lactobacillus plantarum (10%)+Staphylococcus carnosus (90%)), one with lipase and one with both starter culture and lipase were produced in a pilot plant. A lower amount of Micrococcaceae was found in sausages without starter culture. Total FFA quantity at the 15th day was higher in sausages with lipase. Higher amounts of acetic, propionic and butyric acids were found in sausages with both lipase and starter culture. Odor intensity had slightly higher scores in sausages with added lipase.     

黑曲霉脂肪酶在反胶束体系中的光学特性及其催化合成己酸乙酯

研究黑曲霉脂肪酶在反胶束体系中的紫外-可见吸收光谱和荧光光谱特性,并优化黑曲霉脂肪酶在反胶束中催化合成己酸乙酯的反应条件,最后分离和鉴定产物。结果表明：在反胶束体系中黑曲霉脂肪酶的结构发生了变化,芳香族氨基酸暴露更多。黑曲霉脂肪酶催化合成己酸乙酯的反应条件为：在含有异辛烷-正己醇-水的体积比为60:4:1,十六烷基三甲基溴化铵(CTAB)添加量100mmol/L的反胶束体系中,脂肪酶质量浓度为0.003g/L,己酸浓度为0.3mol/L,酸醇物质的量比为1:0.9,反应温度为35℃,摇床转速为120r/min条件下16h时己酸乙酯的合成量达到最大,己酸的转化率达到(88.92±1.00)%。     

Biochemical characterisation and application of lipases produced by Aspergillus sp. on solid‐state fermentation using three substrates

Lipase from Aspergillus sp. obtained by solid‐state fermentation (SSF) on wheat bran (LWB), soybean bran (LSB) and soybean bran combined with sugarcane bagasse (LSBBC) were 67.5, 58 and 57.3 U of crude lipase per gram substrate, respectively. The optimum pH of activity and stability of the LWB was between 8 and 9, and the optimum temperature of activity and stability was 50 °C and up to 60 °C, respectively. The LSB and LSBBC showed two peaks of optimum pH (4 and 6) and optimal values of temperature and stability at 50 °C. The LSB was stable in the pH range of 6–7, while LSBBC in the range of pH 4–7. All the enzymes show activities on p‐nitrophenyl esters (butyrate, laurate and palmitate). LWB stood out either on the hydrolysis of sunflower oil, presenting 66.1% of the activity over commercial lipase and on the esterification of oleic acid and ethanol, surpassing the activities of the commercial lipases studied. The thin layer chromatography showed that LWB and LSB have produced ethyl esters from corn oil, while LWB produced it from sunflower oil.     

米曲霉脂肪酶富集浓缩裂殖壶藻油脂中DHA的机理研究

本研究采用了米曲霉(Aspergillus oryzae, AO)脂肪酶水解法对裂殖壶藻油脂中的二十二碳六烯酸(Docosahexaenoic Acid, DHA)进行了浓缩。在AO脂肪酶最适反应条件下,水解12 h后,反应达到了平衡,产物中甘三酯含量降至40.2%,DHA含量升至55.43%,浓缩了1.58倍,回收率高达68.2%。通过气相色谱结合核磁共振碳谱(13C-NMR)对产物中甘油三酯脂肪酸组成及含量进行了分析,结果显示在酶解反应过程中,DHA倾向于留在甘三酯甘油骨架上,且sn-1,3位上的饱和脂肪酸易被水解而含量下降,表明AO脂肪酶水解反应对甘油骨架上的脂肪酸具有选择性,可将DHA富集在甘油三酯中。AO脂肪酶价格低廉,基于酶解反应的DHA富集工艺简单,深入了解反应的作用机制,将为后续的理论研究和实际应用提供依据。     

南极假丝酵母脂肪酶B在黑曲霉中的分泌表达及其硅藻土固定化应用

为提高南极假丝酵母脂肪酶B（Candida antarctica lipase B,CALB）表达量,根据黑曲霉（Aspergillus niger）密码子偏好性设计合成CALB基因,以PnaII/tpi为启动子构建CALB表达载体并转化到黑曲霉HL-1中表达,摇瓶发酵120 h后上清液中重组CALB活力为171 U/mL。研究重组CALB的酶学性质,最适反应温度和pH值分别为50 ℃和8.0,在pH 6.0～9.0和45 ℃以下具有较高的稳定性,10 mmol/L Cu2＋、Zn2＋和0.1 g/100 mL十二烷基硫酸钠强烈抑制酶活力,而1 mmol/L Ca2＋、0.1 g/100 mL山梨醇对酶活力具有非常明显的激活作用。此外,以硅藻土为载体固定CALB,固定化酶活力为187 U/g。将制备的硅藻土-CALB固定化酶用于生物合成己酸乙酯,经反应条件优化后在无溶剂体系中己酸乙酯产率达91%。     

Changes in enzyme activities and aflatoxin elaboration in sorghum genotypes following Aspergillus parasiticus infestation

Sorghum is a relatively poor substrate for aflatoxin production compared with high‐risk agricultural commodities like maize and groundnut, even though it is susceptible to fungal attack. Fungal infestation of sorghum results in a varied biochemical composition of the deteriorated grain. In this study, six sorghum genotypes (red—AON 486, IS 620; yellow—LPJ, IS 17 779; white—SPV 86, SPV 462) were inoculated with a toxigenic strain of Aspergillus parasiticus (NRRL 2999) in order to evaluate the changes in the activities of various hydrolytic enzymes (α‐ and β‐amylases, protease and lipase) in comparison with those in uninfected grains. Enzyme activities were measured at different times after fungal infestation, and the enzymatic activities were correlated with the aflatoxin production. Alpha‐amylase activity was observed to be greater than β‐amylase activity in all six genotypes under both healthy and infected conditions. The increase in α‐amylase activity during the period of infection was higher in white genotypes than in red sorghum genotypes. Alpha‐amylase activity in all the genotypes increased up to day 6 after fungal infection, but was significantly lower in infected grains than in healthy grains. The variability in the basal enzyme activities among the six sorghum genotypes was quite high compared with the amount of induction of each specific enzyme due to infection and germination. Higher protease activity was observed in the infected grains than in healthy grains. The enzyme activities in high tannin red genotypes were less than those in yellow and white genotypes. The α‐ and β‐amylase activities were positively correlated (r = 0.406 and 0.436; P < 0.05) to aflatoxin production. Inherent lipase activity was highest (on day 0) in AON 486, SPV 462 and SPV 86, as compared with the activity in infected grains. The total aflatoxins produced (quantified by TLC‐fluorodensitometry) were lower in red genotypes than in yellow and white genotypes, suggesting that red genotypes were least susceptible to aflatoxin elaboration among the various genotypes tested. All four aflatoxins, (B₁, B₂, G₁ and G₂) were present in five genotypes (IS 620, LPJ, IS 17 779, SPV 86 and SPV 462) at all the stages of infection, but, aflatoxin could not be detected in the red genotype AON 486 on day 3 after infection. White genotypes SPV 86 and SPV 462) showed maximal aflatoxin (total) production on day 6 after infection. © 2000 Society of Chemical Industry     

脂肪酶固定化新材料

脂肪酶因其在油脂工业中的重要作用而成为酶研究领域的重点。脂肪酶的不稳定性和高昂的价格是限制其广泛应用的主要原因。对脂肪酶进行固定化是增强其稳定性、降低应用成本的关键。改进脂肪酶固定化工艺的一个重要方面就是选择和开发适合相应脂肪酶的固定化材料。从脂肪酶的固定化材料方面,对无机、有机、复合载体等固定化材料进行了综述,特别是对各种新型的固定化材料予以了归纳,对脂肪酶固定化材料的选择有一定的指导意义。     

Overexpression of Aspergillus aculeatus cellobiohydrolase I in Aspergillus oryzae

To express the cbhI gene, encoding Aspergillus aculeatus cellobiohydrolase I (CBHI), in Aspergillus oryzae, a plasmid was constructed. The strain that displayed the strongest CBHI activity among the transformants produced about 941 mg/l in liquid culture. It was confirmed by a PCR method that the plasmid was integrated at the niaD locus.     

醋酸纤维素-聚四氟乙烯复合膜固定化脂肪酶的研究

以醋酸纤维素(CA)和聚四氟乙烯(PTFE)为材料制备醋酸纤维素-聚四氟乙烯复合膜,采用吸附-交联相结合的固定化方法,用该复合膜固定化脂肪酶。研究温度、吸附时间、酶液质量浓度、交联时间和交联剂体积分数对脂肪酶固定化效率和催化效果的影响,并对固定化酶膜的酶学性质进行研究。得到最佳的固定化条件为：温度25℃、吸附时间2h、酶液质量浓度0.02g/mL、交联时间3h、交联剂(戊二醛)体积分数0.2%,固定化酶最大酶活力为17.2U/cm2。固定化酶膜的酶学性质为：最适温度35℃,比游离酶降低了5℃;最适pH8.5,与游离酶相比pH值向碱性偏移1.0;经10次(10h /次)重复使用后,固定化酶相对酶活力为55.5%。SEM结果显示CA-PTFE复合膜能较好的固定化脂肪酶。     

Active lactonizing lipase (LipL) efficiently overproduced by Pseudomonas strains as heterologous expression hosts

Pseudomonas sp. strain 109 secretes lactonizing lipase (LipL), which catalyzes efficient intramolecular transesterification of omega-hydroxyfatty acid esters to form macrocyclic lactones. Because Escherichia coli was found to be unsuitable as an expression host due to the predominant formation of inactive LipL-inclusion bodies and a lack of proper secretion machinery which is also required for the formation of active LipL, Pseudomonas strains were surveyed as expression hosts. Pseudomonas sp. strain 109, an original LipL producer, showed a 7.1-fold higher level of active LipL when the lipL gene under the control of tac-lacUV5 tandem promoter was introduced together with a limL gene encoding a LipL-specific chaperon. Pseudomonas aeruginosa ADD 1976 containing a T7 RNA polymerase gene in the chromosome and plasmid-borne lipL-limL genes under the control of T7 promoter showed a 13-fold higher level of active LipL. Several combinations in the number of lipL and/or limL genes on the plasmid were investigated, and (lipL)3-limL was found to be most efficient, yielding a 67-fold greater production of active LipL than that obtained by the wild-type Pseudomonas sp. strain 109.     

根霉ZZ-3脂肪酶发酵条件的优化及酶学性质研究

对根霉ZZ-3脂肪酶产酶条件的优化及脂肪酶酶学性质进行了研究。结果表明,250 mL三角瓶液体培养,培养基装液量100 mL,根霉ZZ-3产脂肪酶的最佳培养基为：黄豆粉3 g/100mL,（NH4）2S040.3 g/100mL,MgS04 0.2 g/100mL,K2HPO4 0.2 g/100mL,花生油0.3 g/100mL,pH自然。在温度28℃、转速150 r/min,摇床培养时间2 d,发酵酶活力达到124.60 U/mL。对该酶的酶学性质进行了初步研究,该酶最适pH7.5,最适反应温度37℃,在pH7～9、温度低于40℃条件下酶活稳定。     

Regulation of lipid droplet-associated proteins following growth hormone administration and feed restriction in lactating Holstein cows

Lipid metabolism plays a crucial role in the adaptation of dairy cows to periods of energy insufficiency. The objective of the current study was to determine if lipolytic proteins are consistently regulated when energy mobilization is stimulated by different factors. We evaluated 2 models of altered energy balance in mid-lactation Holstein cows, including feed restriction (FR) and administration of bovine growth hormone (GH), by quantifying the abundance and (or) phosphorylation of hormone-sensitive lipase (HSL), perilipin (PLIN), and adipose triglyceride lipase (ATGL). For GH administration, adipose tissue and blood samples were collected 4 d before and 3 and 7 d after administration of GH (n = 20 cows). Similarly, adipose and blood samples were obtained 6 d before and 1 and 4 d after initiation of FR (n = 18 cows). Estimated net energy balance decreased and nonesterified fatty acid concentration increased in both experimental models. Decreased ATGL and PLIN protein abundance was observed with GH administration and FR. Additionally, the abundance of phosphorylated HSL_Ser565 decreased in both models. Decreased abundance of phosphorylated PLIN was observed with GH administration, but not FR. Decreased ATGL protein abundance appears to be a consistent response to energy insufficiency in lactating cows, as this response was also described with negative energy balance at the onset of lactation. In contrast, the abundance of PLIN protein and phosphorylation of HSL using antibodies targeting serine residue 565 of HSL (HSL_Ser565) were altered in the current research, but not at the onset of lactation. Our findings demonstrate that lipolysis is altered through the regulation of multiple proteins, and that this regulation differs according to physiological state in lactating cows.