基于生物酶法的酰基化桑椹花青素的制备与特性

为尽可能减少花青素的降解,提高其稳定性,采用生物酶法,对桑椹花青素进行酰基化修饰,并与非酰基化花青素进行对比研究。运用单因素试验筛选脂肪酶、反应溶剂和酰基供体,分析对花青素酰基转化率的影响。确定酰基化反应的优化条件为南极假丝酵母脂肪酶为酰基化催化酶、吡啶为催化反应溶剂、苯甲酸甲酯为酰基供体,酰基化效果最好,转化率最大为13.5%。采用傅里叶红外、高效液相色谱对产物进行分析,经鉴定,酰基化产物为单酰基或多酰基花青素。此外,研究了酰基化对花青素稳定性和抗氧化性的影响。酰基化可提高花青素的热稳定性、光稳定性和耐酸碱稳定性。相同温度下,酰基化花青素保留率提高约5.0%,光照6d后,酰基化花青素的保留率仍高达96.1%。酰基化可以显著增加花青素体外抗氧化性,增强DPPH自由基清除能力,总还原能力比非酰基化花青素提高30%,金属离子螯合能力高达90%。酰基化花青素肿瘤细胞活性抑制率可达81%,而非酰基化花青素仅为50%。该研究为花青素稳定性提高和性能改善提供参考的理论依据。     

热带假丝酵母产生子囊孢子的条件及单倍体分离的研究

本文研究了诱导热带假丝酵母子囊孢子产生和子囊破壁的条件,以及单倍体分离和鉴别方法。结果表明：NaAc、KCl这两种成分即可满足热带假丝酵母的产孢营养要求,在含有NaAc8．2gm、KCl1．8gm的琼脂培养基中,28℃培养7d,热带假丝酵母细胞的产孢率可达到47．5％;单纯使用蜗牛酶对破除热带假丝酵母子囊壁的效率甚低,酶解液加入适量的石英砂同时振荡处理4h左右,可以显著提高对热带假丝酵母子囊破壁速率。用这种方法处理,绝大多数子囊壁均可破除。子囊破壁处理后再以52℃处理10min杀死残余的营养体细胞,分离物的单倍体孢子比例可由灭活处理前的48％,提高到76％以上。     

马铃薯淀粉渣生料多菌种固态发酵生产蛋白饲料工艺

该研究以提高马铃薯淀粉渣的营养价值为目的。以马铃薯渣为主要原料,麸皮和蚕豆秸秆粉等为辅料,混合生料用纤维素酶进行降解处理后,再经黑曲霉与康宁木霉制备的糖化菌剂糖化降解,最后选用白地霉、产朊假丝酵母、酿酒酵母3种SCP发酵菌种,采用多菌协生生料固态发酵工艺研制马铃薯渣菌体蛋白饲料。通过对发酵产物中真蛋白、粗淀粉等指标的定量分析和感官特征的比较。结果表明,纤维素酶处理后的原料,经黑曲霉与康宁木霉双菌糖化降解后,在28℃条件下,经3种菌白地霉：产朊假丝酵母：酿酒酵母按8∶1.5∶0.5比例接种,混合发酵55 h     

竹黄无性型菌株液体发酵菌丝提取物的生物活性研究

测定竹黄无性型菌株ZHLS-03发酵菌丝体中竹红菌甲素的含量,并对其菌丝提取物的清除自由基活性和抑菌活性进行了研究。按50μg/ml的浓度测定了菌丝体石油醚、乙酸乙酯和甲醇等3种提取物对水稻纹枯病菌、稻瘟病菌、棉花枯萎病菌3种植物病原菌以及白色假丝酵母、金黄色葡萄球菌2种病原细菌的抑制能力;同时测定菌丝体的石油醚、乙酸乙酯和饱和正丁醇等3种提取物对DPPH自由基的清除率。结果表明：菌株ZHLS-03液体培养至6d,菌丝体中竹红菌甲素含量最高,可达到28029mg/g;菌丝体甲醇和正丁醇提取物分别具有较强的抑菌活性和清除自由基活性,甲醇相对棉花枯萎病菌的抑制率为7478%,抗白色假丝酵母的抑菌圈直径达到2069mm,且反应15min后正丁醇提取物对DPPH自由基的清除率可达9324%。     

外源蛋白在巴斯德毕赤酵母中高效分泌表达的前沿技术

巴斯德毕赤酵母表达系统是目前应用非常广泛的外源蛋白真核表达系统。外源蛋白在巴斯德毕赤酵母中的高效分泌表达有利于减少下游纯化步骤,降低生产成本,具有重大的实用意义。本文综述了生物信息学、系统生物学、高通量筛选和合成生物学等前沿技术在信号肽和代谢系统优化中的应用,为进一步提高外源蛋白在巴斯德毕赤酵母中的分泌表达提供参考。     

锰超氧化物歧化酶（Mn-SOD）基因的分子改造及在毕赤酵母中的表达

根据毕赤酵母（Pichia pastoris）的密码子偏好性,以不改变氨基酸序列为原则,对源于蜡样芽孢杆菌M22（Bacillus cerues M22）的Mn-SOD基因进行分子改造,设计、合成新的基因序列Mn-SOD-2。构建酵母表达载体pPICZαA/Mn-SOD-2,并整合至毕赤酵母GS115染色体。结果表明,所构建的酵母工程菌株YM103,经0.5%甲醇诱导表达后,Native-PAGE检测证实有清晰活性条带;SDS-PAGE检测证实重组蛋白的分子量为24kD,与预期大小一致。酶活分析表明,外源蛋白的活性较改造前增加了2.2倍,且表达稳定性良好。     

蜡样芽胞杆菌M22Mn-SOD基因的分子改造及在毕赤酵母中的表达

根据毕赤酵母(Pichia pastons)密码子的偏好性,以氨基酸序列不变为原则,对源于蜡样芽胞杆菌(Bacillus cernes)M22的Mn-SOD基因进行分子改造,设计、合成了新的基因序列Mn-SOD-2.构建酵母表达载体pPICZαA/Mn-SOD-2,并整合至毕赤酵母GS115染色体.结果表明,所构建的重组体经0.5%甲醇诱导表达后,Native-PAGE检测证实有清晰单一活性条带;SDS-PAGE检测证实重组蛋白的分子量24 kD.酶活分析表明,外源蛋白的活性较改造前增加了2.2倍,且表达稳定性良好.     

响应面法优化制备高富硒产朊假丝酵母

考察了发酵条件对产朊假丝酵母富硒能力的影响。通过单因素的筛选,对酵母富硒能力影响较大的3个因素:亚硒酸钠浓度、初始p H值及培养温度,以胞内总硒含量为响应值,利用响应面法对其进行优化。结果显示:在培养时间30 h、加硒时间对数生长中期、亚硒酸钠浓度35 mg·L-1,初始p H 6.6、接种量10%、培养温度27℃、装液量150 m L/500 m L的条件下,最大的菌体生物量为6.87 g·L-1;胞内总硒含量达到12 639.7μg·L-1,硒含量为1 839.8μg·g-1,其中亚硒酸钠转化率为79.1%,有机硒含量占90%以上;胞内实际总硒含量与数学模型理论值12 518.8μg·L-1相差不显著,响应面法能较好地优化产朊假丝酵母富硒工艺条件。     

甜高粱茎秆残渣生料多菌种固态发酵生产蛋白饲料

为实现甜高粱茎秆残渣生产蛋白饲料的规模化应用,该研究将黑曲霉(Aspergillus niger)、里氏木霉(Trichoderma reesei),产朊假丝酵母(Candida utilis)和干酪乳杆菌(Lactobacillus casei)进行优化组合,添加不同质量分数尿素对甜高粱茎秆残渣进行生料固态发酵生产蛋白饲料。试验通过对比发酵前后粗蛋白、真蛋白、粗灰分和粗脂肪质量分数变化发现:添加4种菌的复合菌株,再添加1%尿素发酵8 d后能使以甜高粱茎秆残渣为底物的饲料中纤维素由33.00%降低至24.09%,半纤维素由20.99%降低至17.69%;粗蛋白质量分数由2.27%提升至7.14%,真蛋白由2.01%提升至6.41%。该研究简化了甜高粱秸秆残渣规模化生产饲料蛋白的工艺,为该工艺的推广应用奠定基础。     

基于CRISPR系统基因编辑与基因调控技术的发展

由规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)以及CRISPR相关蛋白9(CRISPR-associated protein 9,Cas9)组成的系统被发现广泛存在于细菌及古菌中,是机体长期进化形成的由RNA指导的降解外源遗传物质的适应性免疫系统。由于该系统可以识别靶向序列完成DNA的双链切割,因此从2013年起,CRISPR/Cas9系统即被改造为基因编辑工具。作为一种新型的基因组编辑技术,CRISPR/Cas9系统具有设计简单、特异性强、效率高等优点,为基因组定向改造调控和应用带来了突破性的革命,并且迅速在基础理论、转基因动物生产、基因诊断和临床治疗等领域中得到广泛的研究与应用。然而,CRISPR/Cas9也存在着脱靶效应和外源基因插入困难等一些亟待解决的问题,这在一定程度上限制了CRISPR/Cas9的应用。因此,近年来围绕CRISPR系统改进获得了一系列可用于基因编辑与基因表达修饰的新工具。本文介绍了CRISPR系统的组成、工作原理,并综述了近几年来基于CRISPR系统开发的几种新型基因编辑工具以及基因表达修饰工具的研究进展和应用,并对其应用前景和发展方向进行了展望。     

Highly efficient preparation of lipophilic hydroxycinnamates by solvent-free lipase-catalyzed transesterification

Various medium- or long-chain alkyl cinnamates and hydroxycinnamates, including oleyl p-coumarate as well as palmityl and oleyl ferulates, were prepared in high yield by lipase-catalyzed transesterification of an equimolar mixture of a short-chain alkyl cinnamate and a fatty alcohol such as lauryl, palmityl, and oleyl alcohol under partial vacuum at moderate temperature in the absence of solvents and drying agents in direct contact with the reaction mixture. Immobilized lipase B from Candida antarctica was the most effective biocatalyst for the various transesterification reactions. Transesterification activity of this enzyme was up to 56-fold higher than esterification activity for the preparation of medium- and long-chain alkyl ferulates. The relative transesterification activities found for C. antarctica lipase were of the following order: hydrocinnamate > cinnamate > 4-hydroxyhydrocinnamate > 3-methoxycinnamate > 2-methoxycinnamate approximately 4-methoxycinnamate approximately 3-hydroxycinnamate > hydrocaffeate approximately 4-hydroxycinnamate > ferulate > 2-hydroxycinnamate > caffeate approximately sinapate. With respect to the position of the hydroxy substituents at the phenyl moiety, the transesterification activity of C. antarctica lipase B increased in the order meta > para > ortho. The immobilized lipases from Rhizomucor miehei and Thermomyces lanuginosus demonstrated moderate and low transesterification activity, respectively. Compounds with inverse chemical structure, that is, 3-phenylpropyl alkanoates such as 3-(4-hydroxyphenyl)propyl oleate and 3-(3,4-dimethoxyphenyl)propyl oleate, were obtained by C. antarctica lipase-catalyzed transesterification of fatty acid methyl esters with the corresponding 3-phenylpropan-1-ols in high yield, as well.     

High-yield preparation of wax esters via lipase-catalyzed esterification using fatty acids and alcohols from crambe and camelina oils

Fatty acids obtained from seed oils of crambe (Crambe abyssinica) and camelina (Camelina sativa) via alkaline saponification or steam splitting were esterified using lipases as biocatalysts with oleyl alcohol and the alcohols derived from crambe and camelina oils via hydrogenolysis of their methyl esters. Long-chain wax esters were thus obtained in high yields when Novozym 435 (immobilized lipase B from Candida antarctica) and papaya (Carica papaya) latex lipase were used as biocatalysts and vacuum was applied to remove the water formed. The highest conversions to wax esters were obtained with Novozym 435 (> or =95%) after 4-6 h of reaction, whereas with papaya latex lipase such a high degree of conversion was attained after 24 h. Products obtained from stoichiometric amounts of substrates were almost exclusively (>95%) composed of wax esters having compositions approaching that of jojoba (Simmondsia chinensis) oil, especially when crambe fatty acids in combination with camelina alcohols or camelina fatty acids in combination with crambe alcohols were used as substrates.     

Lipase-catalyzed synthesis of fatty acid diethanolamides.

Diethanolamides are nonionic emulsifiers widely used in industries such as cosmetics and as corrosion inhibitors. Candida antarctica lipase (Novozym 435) was used to catalyze the amidation of various fatty acids with diethanolamine. Contents of fatty acids, metal ions, and water affected the yields of diethanolamides. Hexanoic acid was the best substrate among all acyl donors. Yields of hexanoyl diethanolamide (HADEA), lauroyl diethanolamide (LADEA), and oleoyl diethanolamide (OADEA), obtained after 24 h of lipase-catalyzed reaction at 50 degrees C and 250 rpm with 90 mM fatty acid and 360 mM diethanolamine in acetonitrile, were 76.5, 49.5, and 12.1%, respectively. Addition of 1 mM metal salts increased the yields of HADEA and LADEA. Kinetic analysis showed that the yields of HADEA and LADEA in lipase-catalyzed reactions were largely associated with the rate of the forward reaction constant k(1). Anhydrous enzyme was found to be the best for the amidation reaction. Study on the enzyme operational stability showed that C. antarctica lipase retained 95 and 85% of the initial activity for the syntheses of HADEA and LADEA, respectively (even after repeated use for 10 days). The reaction runs smoothly without the use of hazardous reactants, and the developed method is useful for the industrial application.     

Process development of continuous glycerolysis in an immobilized enzyme-packed reactor for industrial monoacylglycerol production

Continuous and easily operated glycerolysis was studied in different lipase-packed columns to evaluate the most potential process set-ups for industrial monoacylglycerol (MAG) production. Practical design-related issues such as enzyme-filling degree, required reaction time, mass transfer investigations, and capacity and stability of the enzyme were evaluated. A commercially available immobilized Candida antarctica lipase B was used to catalyze the glycerolysis reaction between glycerol and sunflower oil dissolved in a binary tert-butanol: tert-pentanol medium. Considering easy handling of the enzyme and measured expansion when wetted with a reaction mixture, a filling degree of 52 vol % dry enzymes particles per column volume seemed appropriate. Twenty minutes was required to reach equilibrium conditions with a MAG content of 50-55 wt %. Only insignificant indications of mass transfer limitations were observed. Hence, the commercial lipase seemed adequate to use in its available particle size distribution ranging from 300 to 900 microm. A column length-to-diameter ratio of less than 25 did not interfere with the transfer of the fluid mixture through the column. Under the tested conditions, the enzyme could be active for approximately 92 days before enzyme renewal was needed. This corresponds to a very high enzyme capacity with approximately 2000 L pure MAG produced per kg enzyme.     

Stereoselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol by lipase-catalyzed hydrolysis of 3-acetylthioesters

The enantioselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol, two potent sulfur-containing aroma compounds, by lipase-catalyzed hydrolysis of the corresponding 3-acetylthioesters was investigated. The stereochemical course of the kinetic resolutions was followed by capillary gas chromatography using modified cyclodextrins as chiral stationary phases. The enzyme preparations tested varied significantly in terms of activity and enantioselectivity (E). The highest E value (E = 36) was observed for the hydrolysis of 3-acetylthiohexanal catalyzed by lipase B from Candida antarctica resulting in (S)-configured thiol products. Immobilization of the enzyme (E = 85) and the use of tert-butyl alcohol as cosolvent (E = 49) improved the enantioselectivity. Modification of the acyl moiety of the substrate (3-benzoylthiohexanal) had no significant impact. The sulfur-containing compounds investigated possess attractive odor properties, and only one of the enantiomers exhibits the pleasant citrus type note.     

Acidolysis of tristearin with selected long-chain fatty acids

Five lipases, namely, Candida antarctica (Novozyme-435), Mucor miehei (Lipozyme-IM), Pseudomonas sp. (PS-30), Aspergillus niger (AP-12), and Candida rugosa (AY-30), were screened for their effect on catalyzing the acidolysis of tristearin with selected long-chain fatty acids. Among the lipases tested C. antarctica lipase catalyzed the highest incorporation of oleic acid (OA, 58.2%), gamma-linolenic acid (GLA, 55.9%), eicosapentaenoic acid (EPA, 81.6%), and docosahexaenoic acid (DHA, 47.7%) into tristearin. In comparison with other lipases examined, C. rugosa lipase catalyzed the highest incorporation of linoleic acid (LA, 75.8%), alpha-linolenic acid (ALA, 74.8%), and conjugated linoleic acid (CLA, 53.5%) into tristearin. Thus, these two lipases might be considered promising biocatalysts for acidolysis of tristearin with selected long-chain fatty acids. EPA was better incorporated into tristearin than DHA using the fifth enzymes. LA incorporation was better than CLA. ALA was more reactive than GLA during acidolysis, except for the reaction catalyzed by Pseudomonas sp., possibly due to structural differences (location and geometry of double bonds) between the two fatty acids. In another set of experiments, a combination of equimolar quantities of unsaturated C18 fatty acids (OA + LA + CLA + GLA + ALA) was used for acidolysis of tristearin to C18 fatty acids at ratios of 1:1, 1:2, and 1:3. All lipases tested catalyzed incorporation of OA and LA into tristearin except for M. miehei, which incorportaed only OA. C. rugosa lipase better catalyzed incorporation of OA and LA into tristearin than other lipases tested, whereas the lowest incorporation was obtained using Pseudomonas sp. As the mole ratio of substrates increased from 1 to 3, incorporation of OA and LA increased except for the reaction catalyzed by A. niger and C. rugosa. All lipases tested failed to allow GLA or CLA to participate in the acidolysis reaction, and ALA was only slightly incoporated into tristearin when M. miehei was used.     

Solvent-free lipase-catalyzed preparation of long-chain alkyl phenylpropanoates and phenylpropyl alkanoates

An enzymatic method was developed for the preparation of medium- or long-chain alkyl 3-phenylpropenoates (alkyl cinnamates), particularly alkyl hydroxy- and methoxy-substituted cinnamates such as oleyl p-coumarate and oleyl ferulate. The various alkyl cinnamates were formed in high to moderate yield by lipase-catalyzed esterification of cinnamic acid and its analogues with fatty alcohols in vacuo at moderate temperatures in the absence of drying agents and solvents. Immobilized Candida antarctica lipase B was the most effective biocatalyst for the various esterification reactions. The relative esterification activities were of the following order: dihydrocinnamic > cinnamic > 3-methoxycinnamic > dihydrocaffeic approximately 3-hydroxycinnamic > 4-methoxycinnamic > 2-methoxycinnamic > 4-hydroxycinnamic > ferulic approximately 3,4-dimethoxycinnamic > 2-hydroxycinnamic acid. With respect to the position of the substituents at the phenyl moiety, the esterification activity increased in the order meta > para > ortho. Rhizomucor miehei lipase demonstrated moderate esterification activity. Compounds with inverse chemical structure, that is, 3-phenylpropyl alkanoates such as 3-(4-hydroxyphenyl)propyl oleate, were also obtained in high yield by esterification of fatty acids with the corresponding 3-phenylpropan-1-ols.     

Solvent-free lipase-catalyzed preparation of diacylglycerols

Various methods have been applied for the enzymatic preparation of diacylglycerols that are used as dietary oils for weight reduction in obesity and related disorders. Interesterification of rapeseed oil triacylglycerols with commercial preparations of monoacylglycerols, such as Monomuls 90-O18, Mulgaprime 90, and Nutrisoft 55, catalyzed by immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) in vacuo at 60 degrees C led to extensive (from 60 to 75%) formation of diacylglycerols. Esterification of rapeseed oil fatty acids with Nutrisoft, catalyzed by Lipozyme RM in vacuo at 60 degrees C, also led to extensive (from 60 to 70%) formation of diacylglycerols. Esterification of rapeseed oil fatty acids with glycerol in vacuo at 60 degrees C, catalyzed by Lipozyme RM and lipases from Thermomyces lanuginosus (Lipozyme TL IM) and Candida antarctica (lipase B, Novozym 435), also provided diacylglycerols, however, to a lower extent (40-45%). Glycerolysis of rapeseed oil triacylglycerols with glycerol in vacuo at 60 degrees C, catalyzed by Lipozyme TL and Novozym 435, led to diacylglycerols to the extent of 相似文献   

On-line supercritical fluid extraction/enzymatic hydrolysis of vitamin a esters: a new simplified approach for the determination of vitamins a and e in food

An on-line supercritical fluid extraction (SFE)/enzymatic hydrolysis procedure using immobilized lipase has been developed for the determination of vitamin A in dairy and meat products. Several lipases were tried, of which Novozyme 435 (Candida antarctica type B) showed the highest activity toward retinyl palmitate. There was no observed activity with alpha-tocopheryl acetate. When pressure, temperature, modifiers, flow rate, extraction time, and water content were varied, high vitamin A recovery was obtained in milk powder. Collected extracts were analyzed by reversed-phase high-performance liquid chromatography with ultraviolet and fluorescence detection without additional sample cleanup. The procedure gave reliable values of vitamin A as well as of vitamin E in other food items such as infant formula, minced pork and beef meat, and low- and high-fat liver paste. The described method is faster and more automated than conventional methods based on liquid-liquid extraction, or SFE using off-line saponification, for vitamin A and E determination. Results obtained with the new method did not differ significantly from those obtained with the other two methods mentioned above.     

Solvent-free enzymatic preparation of feruloylated monoacylglycerols optimized by response surface methodology

The ability of immobilized lipase B from Candida antarctica (Novozym 435) to catalyze the direct esterification of glyceryl ferulate (FG) and oleic acid for feruloylated monoacylglycerols (FMAG) preparation in a solvent-free system was investigated. Enzyme screening and the effect of glycerol on the initial reaction rate of esterification were also investigated. Response surface methodology (RSM) was used to optimize the effects of the reaction temperature (55-65 degrees C), the enzyme load (8-14%; relative to the weight of total substrates), oleic acid/(FG + glycerol) (6:1-9:1; w/w), and the reaction time (1-2 h) on the conversion of FG and yield of FMAG. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of FG conversion and FMAG yield. The optimum preparation conditions were as follows: temperature, 60 degrees C; enzyme load, 8.2%; substrate ratio, 8.65:1 (oleic acid/(FG + glycerol), w/w); and reaction time, 1.8 h. Under these conditions, the conversion of FG and yield of FMAG are 96.7 +/- 1.0% and 87.6 +/- 1.2%, respectively.