脂肪酶热稳定性改造研究进展

热稳定性改造是脂肪酶酶学性质研究的热点之一,随着技术的发展,越来越多的有效手段被用于脂肪酶热稳定性改造。作者综述了影响脂肪酶热稳定性的主要因素、提高其热稳定性的研究方法以及最新研究成果,为脂肪酶的进一步改造提供依据,并对其他酶的酶学性质改造具有借鉴意义。     

基于易错PCR技术的α-淀粉酶基因的定向进化研究

采用基因体外定向进化策略中易错PCR技术,用碱基类似物5-溴脱氧尿苷三磷酸(5-BrdUTP)部分取代脱氧胸苷三磷酸(dTTP),对野油菜黄单胞菌的α-淀粉酶基因进行了体外诱变.通过鉴别培养基进行筛选,得到5个具有高酶活的诱变基因,利用生物学软件DNAstar和出发基因序列进行比较,分析了突变位点和酶功能变化的相应关系.结果显示:基因诱变后酶活的改变主要是由淀粉酶基因空间结构的改变向引起的,而不是由启动子的变化引起的.     

In vitro immunogenicity of various native and thermally processed bovine milk proteins and their mixtures

In vitro immunogenicity of various native and thermally processed (72°C/15 s and 100°C/30 s) bovine milk protein fractions, their mixtures, whey, and skim milk, was studied by analyzing the immune response of T helper (Th) cells in human peripheral blood mononuclear cells. The secretion of Th type cytokines induced by the protein stimulants was quantified while determining the heat-induced protein denaturation. Purified whey proteins, caseins and whey fraction, and skim milk provoked substantial immune responses at various degrees, indicating their potent immunogenicity. The protein mixtures prepared using the fractionated whey proteins with or without caseins appeared less immunogenic in both native and heat-treated forms, implying their potential of producing less immunogenic dairy products. The 100°C/30 s treatment significantly altered the immunogenicity of most of the potent protein stimulants, which mostly coincided with their levels of protein denaturation. The 72°C/15 s treatment caused the least protein denaturation but altered the immunogenicity of several protein stimulants notably, including heat-stable caseins and α-lactalbumin.     

Introduction of sulphydryl groups into pea vicilin: Formation of intra-and inter-polypeptide disulphide bonds

Site directed mutagenesis was used to introduce two cysteine residues into the hydrophilic regions of the pea vichlin polypeptide previously expressed in Saccharomyces cerevisiae. The mutated polypeptide was expresed and the resultant protein was characterised by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. The presence of both intra- and inter-polypeptide sulphydryl bonds was indicated by bands of different mobility from those of the native polypeptide. The association of mutated vicili polypeptides into disulphide-bonded aggregates within the yeast was not a random process, trimers being the major aggregate produced. When the mutated vicilin was extracted from yeast and purified, random aggregates of the vicilin polypeptide were formed.     

In vitro glycation and antigenicity of soy proteins

Soy protein isolate (SPI) was glycated with fructose or fructooligosaccharides (FOS) through the Maillard reaction in powder and liquid systems. A reduction in primary free amino groups of SPI up to 85% and 96% was observed in the powder and liquid system, respectively. Following heating at 95 °C for 1 h under liquid conditions, the electrophoretic behavior of allergenic 7S (β-conglycinin) and 11S (glycinin) fractions of SPI was modified when glycated with FOS (molar ratio primary amino groups to FOS of 1:74) as shown by SDS-PAGE analysis. The antigenicity of this glycated protein was also largely reduced (up to ∼90%) compared with that of the unglycated form. Glycation reactions with fructose in a powder and liquid system also reduced the antigenicity of the glycated proteins.     

易错PCR技术定向进化褐藻胶裂解酶Alg-2的分析

为提高褐藻胶裂解酶活力,采用易错聚合酶链式反应（polymerase chain reaction,PCR）技术对海洋细菌Tamlana sp. S12中具有代表性的酶组分Alg-2进行体外定向进化。经过2?轮易错PCR及96?孔板发酵筛选,分别获得2?株正突变和2?株负突变菌株,其酶比活力分别是亲本（2?675.2?U/g）的162%、241%、53%、11%。酶的动力学分析显示,正突变株P2-81的Km值比亲本降低50%,而负突变株N2-47的Km值比亲本提高106%,表明突变株对底物的亲和力有极大的上升和下降。基因测序及氨基酸序列分析结果表明,Glu、Thr、Ser、Asp和Tyr对褐藻胶裂解酶活力提高起到正面的积极作用,而Lys的突变起到负面的消极作用,后续人工合成的定点突变则进一步证实Asp和Lys的正/负作用。本研究有助于深入理解褐藻胶裂解酶的催化机制,为后续利用理性设计手段改造褐藻胶裂解酶提供理论参考。     

Directed evolution of angiotensin II-inhibiting peptides using a microbead display

Angiotensin II (ang II), an octapeptide (DRVYVHPF), can regulate blood pressure by binding specifically to its receptor, AT1. A peptide (VVIVIY) in the first transmembrane of AT1 has been found, via peptide array technology, to have an affinity for ang II. In this study, the peptide P2, which contained the VVIVIY sequence, was mutated and screened using microbead display technology that utilized emulsion PCR and cell-free protein synthesis. After one round of screening, the binding activities of collected mutants were estimated using flow cytometry and a peptide array. Two of these exhibited improved association rate constants to ang II, compared to the P2 peptide.     

In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus

An in vivo-directed evolutionary strategy was used to obtain a thermostabilized Escherichia coli hygromycin B phosphotransferase, using a host-vector system of Thermus thermophilus. Introduction of the mutant gene containing two amino acid substitutions, S52T and W238C, which was previously reported by Cannio et al. [J. Bacteriol., 180, 3237-3240 (1998)], did not confer hygromycin resistance on T. thermophilus cells at 55 degrees C; however, five spontaneously-generated independent mutants were obtained by selection of the transformants at this temperature. Each mutant gene contained one amino acid substitution of either A118V or T246A. Further selection with increasing temperature, at 58 degrees C and then 61 degrees C, led to acquisition of three more substitutions: D20G, S225P and Q226L. These mutations cumulatively influenced the maximum growth temperature of the T. thermophilus transformants in the presence of hygromycin; T. thermophilus carrying a mutant gene containing all the five substitutions was able to grow at up to 67 degrees C. This mutant gene, hph5, proved useful as a selection marker in the T. thermophilus host-vector system, either on the plasmid or by genome integration, at temperatures up to 65 degrees C.     

太空诱变复合菌剂发酵苹果渣生产菌体蛋白的研究

试验利用太空诱变获得的优良菌种一黑曲霉ZM-8和啤酒酵母、热带假丝酵母以及白地霉复配制成复合菌剂,固体发酵苹果渣,得到的菌体蛋白中纤维素含量降低,粗蛋白含量显著提高,营养价值增加.通过正交试验获得了最适的培养基配方和复合菌剂的最佳配方.试验确定的最适培养基配方为白糖4g,硫铵7g,尿素1g,食盐2g,产物中粗蛋白的含量达26.4%,提高了306.3%.复合菌剂的最佳配方为黑曲霉突变株ZM-8(Asp.niger ZM-8)1.5g,酿酒酵母(Saccharomyces cerevisiae)0.5g,热带假丝酵母(Candida tropicalis)1.0g,白地霉菌(Gcotrichum candidum)0.5g,产物中粗蛋白的含量达27.9%,提高了337.5%.     

In vitro multienzyme digestibility of protein of six varieties of African yam bean flours

The in vitro multienzyme protein digestibilities of the flours of six varieties of African yam bean (Sphenostylis stenocarpa), made from both whole seeds and dehulled seeds were investigated. The multienzyme system consisted of trypsin, chymotrypsin and peptidase. Digestibilities were determined for a 10 min digestion period. Both dehulling and heat treatment improved digestibility. Comparison of flours from raw whole seeds with those from dehulled seeds showed that digestibility was better in the latter, with an increase of 6.78%. Heat-treated whole seed flcurs gave a digestibility increase of 6.06% compared with raw flours whereas in the dehulled samples the digestibility increase of heat-treated flours over raw was 5.19%. Heat-treated dehulled seeds were better, with a digestibility increase of 5.90% over the heat-treated whole seed flours. No significant differences (P < 0.05) were observed in the digestibilities among the whole seed flours but significant differences were observed in the dehulled seed flours.     

Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo

Rotavirus is a major cause of infantile viral gastroenteritis and can lead to severe and sometimes lethal dehydration. Previous studies have shown that breast-fed children are better protected against symptomatic infections, and that the milk fat globule protein lactadherin might be at least partly responsible for this effect. In vitro studies have shown that human lactadherin, in contrast to the bovine ortholog, could inhibit rotavirus infectivity, and that bovine MUC1 and a commercially available bovine macromolecular whey protein (MMWP) fraction proved to be effective. The present work describes the versatility of MMWP against the infection of 2 human intestinal cell lines (Caco-2 and FHs 74 Int) by 4 different rotavirus strains (Wa, RRV, YM, RF). Isolation of a protein fraction (CM3Q3) from MMWP that effectively inhibits rotavirus infectivity in vitro is documented. Purification was achieved by monitoring the rotaviral inhibitory activity in fractions obtained from 2 consecutive steps of ion-exchange chromatography. The major component of CM3Q3 was shown to be bovine IgG, and the attenuating capacity of this fraction is most properly linked to this component. The capacity of MMWP, MUC1, lactadherin, and the CM3Q3 fraction to inhibit the infectivity of the murine EMcN rotavirus strain was analyzed in adult BALB/c mice by using 2 different amounts of virus (10 and 100 times more than 50% the viral shedding doses). Only CM3Q3 was able to significantly affect the shedding of rotavirus in the stools of experimentally infected mice when the high viral dose was given. Detection of rotavirus-specific serum antibodies showed that the high dose infected all groups of mice. Experiments with the low dose of virus implied that all the tested milk proteins could affect the viral shedding in stools; in addition, use of MUC1, MMWP, and CM3Q3 prevented the appearance of serum viral antibodies. The advantages of using bovine immunoglobulins to induce passive immunity against rotavirus have been substantially investigated, although studies have mainly focused on the use of derivatives from immunized cows, especially colostrum. This report associates considerable activity against rotavirus infectivity with an ordinary whey product, suggesting that there might be alternatives to colostral-derived products.     

In vitro digestibility of protein and amino acids in protein mixtures

Six protein sources, casein, field peas, peanut meal, wheat flour, rapeseed and soya bean concentrate and their blends (ratio 1:1) were subjected to in vitro enzymic digestion. Wheat flour had the lowest in vitro digestibility (30% in 6 h) while the other sources had similar digestibilities (40%). Basic and aromatic amino acids were the most readily liberated from these protein sources. Some protein combinations, such as a rapeseed/field pea blend, gave in vitro digestibilities higher than calculated from individual proteins. The type of response observed could not be predicted from either nitrogen digestibility or amino acid composition of the individual sources. The digestibility of some amino acids was modified and this could be due to varying affinity of digestive enzymes for the protein.     

植物乳杆菌单宁酶的定向进化及应用

通过对植物乳杆菌（Lactobacillus plantarum, L. plantarum)单宁酶（Lptan1）的定向进化可提高其热稳定性和比酶活,进一步研究正向突变体（mLptan1）对绿茶茶汤中酯类儿茶素的降解效果。通过易错PCR构建Lptan1的突变文库,采用高通量筛选耐热性和比酶活提高的突变体并在大肠杆菌中表达,评价其在水解绿茶中酯类儿茶素方面的应用。筛选得到一个mLptan1,其最适反应温度（45 ℃）比Lptan1（40 ℃）提高了5 ℃。比酶活（425.6 U/mg）是Lptan1（312.3 U/mg）的1.36倍。将mLptan1应用于绿茶茶汤,在加酶量1 U/mL和最适反应温度下水解120 min,茶汤中酯类儿茶素降解率达85%,而Lptan1降解率仅为65%。结果表明,定向进化明显提高了Lptan1的最适反应温度和茶汤中酯类儿茶素的降解率,在茶类饮料中具有很好的应用前景。     

基于定向进化技术提高地衣芽孢杆菌L-天冬酰胺酶活性

为提高地衣芽孢杆菌L-天冬酰胺酶活性,通过定向进化技术对其进行分子改造。经过两轮易错聚合酶链式反应和一轮DNA shuffling,从19 100多个突变株中筛选到突变体S10、S16和S21,其酶比活力较野生型分别提高了106%、74%和43%,且突变酶K_(cat)/K_m都有所增大。其中,突变体S10氨基酸序列发生3个突变,K43E、N67S和I269L。三维模拟结果显示,第43位氨基酸突变为谷氨酸、第67位氨基酸突变为丝氨酸,可能提高了底物亲和力和催化效率,从而提高酶活性。圆二色谱分析表明,相比野生酶,突变酶的α-螺旋数减少、无规则卷曲有所增加,表明其刚性略有降低,柔性有所增加。利用定向进化策略能够有效地提高地衣芽孢杆菌L-天冬酰胺酶活性。     

In vitro determination of antioxidant activity of proteins from jellyfish Rhopilema esculentum

In this study, the antioxidant activity of proteins isolated from jellyfish, Rhopilema esculentum Kishinouye (R. esculentum), was determined by various antioxidant assays, including superoxide anion radical-scavenging, hydroxyl radical-scavenging, total antioxidant activity, reducing power and metal chelating activity. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol, vitamin C and mannitol were used as standards in those various antioxidant activities. The crude protein (CP) and the protein fractions isolated by Sephadex chromatography, first peak (FP) and second peak (SP), had very low reductive power and metal chelating abilities compared to EDTA, but they showed strong scavenging effects on the superoxide anion radical, hydroxyl radical and varying total antioxidant activity. FP and SP exhibited stronger scavenging effects on the superoxide anion radical than BHA, BHT or α-tocopherol. The EC₅₀ values of FP and SP were 6.12 and 0.88 μg/ml, respectively, while values EC₅₀ of BHA, BHT and α-tocopherol were 31, 61 and 88 μg/ml, respectively. CP, FP and SP showed far higher hydroxyl radical-scavenging activities than did vitamin C or mannitol. The EC₅₀ values of CP, FP and SP were 48.76, 45.42 and 1.52 μg/ml, but EC₅₀ values of vitamin C and mannitol were 1907 and 4536 μg/ml, respectively. In a β-carotene–linoleate system, SP and CP showed antioxidant activity, but lower than BHA. Of the three samples, SP had the strongest antioxidant activity. So, SP may have a use as a possible supplement in the food and pharmaceutical industries.     

普鲁兰酶N467G突变体的酶学性质分析

在淀粉制糖工业中,普鲁兰酶通常与糖化酶配合使用,其在酸性pH和较高温度下的催化活力是影响淀粉脱支效率的主要因素.该研究通过对长野芽胞杆菌(Bacillus naganoensis)普鲁兰酶蛋白质解折叠自由能的差值(ΔΔG)的计算预测和突变位点稳定性分析,选择突变位点并进行定点突变,获得突变体N467G.与野生型普鲁兰酶...     

基于定向进化技术提高巨大芽孢杆菌谷氨酸脱羧酶活性

为提高巨大芽孢杆菌谷氨酸脱羧酶活性,通过定向进化技术对其进行酶工程改造。经过二轮易错聚合酶链式反应,从13 000 多个突变株中筛选到突变株A5-3、E2-4、E3-11,相对于野生型,其酶比活力提高了157%、115%、97%,且Kcat/Km都有所增大。其中A5-3氨基酸序列发生了2 个突变（A55D和D451E）。三维模拟结果表明,第55位丙氨酸突变为天冬氨酸很可能为酶促反应提供H＋,从而加快酶促反应效率;突变株E2-4第34位由亮氨酸突变成谷氨酰胺,一定程度上改善了酶的热稳定性;突变株E3-11的第325位由丙氨酸突变成丝氨酸有利于蛋白内部形成更多氢键,增大了该部位的柔性,更有利于氨基酸残基之间发生相互作用。圆二色谱分析表明,突变株与野生型具有相似的三维结构,相比于野生酶,突变酶的α-螺旋减少,无规则卷曲增加,说明突变酶刚性有所下降而柔性增加。利用定向进化技术可以明显改善谷氨酸脱羧酶的酶活性,为其工业化的应用提供实验参考。