脑蛋白粉的制备及其酶解方法的研究

研究了以鲜猪脑制备蛋白粉并进行酶水解的方法 ;脑蛋白粉质量、不同酶和加酶方法对水解结果的影响 ;分析了水解液中游离氨基酸和低分子肽谱 ,并与国内外类似研究进行了比较。结果表明 :丙酮、乙醚沉淀制得的脑蛋白粉含氮 9.0 %～ 1 1 .5% ,脂肪 2 .5%～ 4 .0 % ,适于酶解 ;水解液含有 1 6种游离氨基酸 ,总量达 3 2 .4 4mg/ml和 4个分子量小于 1万的肽。提出精制脑蛋白粉进行水解、减压浓缩、超滤是提高水解液有效成分的技术措施。     

脑蛋白粉的制备及其酶解方法的研究

研究了以鲜猪脑制备蛋白粉并进行酶水解的方法 ;脑蛋白粉质量、不同酶和加酶方法对水解结果的影响 ;分析了水解液中游离氨基酸和低分子肽谱 ,并与国内外类似研究进行了比较。结果表明 :丙酮、乙醚沉淀制得的脑蛋白粉含氮 9.0 %～ 1 1 .5% ,脂肪 2 .5%～ 4 .0 % ,适于酶解 ;水解液含有 1 6种游离氨基酸 ,总量达 3 2 .4 4mg/ml和 4个分子量小于 1万的肽。提出精制脑蛋白粉进行水解、减压浓缩、超滤是提高水解液有效成分的技术措施。     

制革醛鞣固体废弃物的酶法资源化利用

为建立毛皮制革产生固体废弃物的清洁化、资源化利用工艺,采用JW酶制剂对兔毛皮甲醛鞣制固废进行酶解处理,并对酶解产物的工农业用途进行评估。结果表明,固废中的皮块能够被JW-4酶制剂完全水解。皮块水解后回收的毛纤维经检测其长度优于刀剪工艺,断裂强度、伸长率与原始纤维相当。酶解液中含大量小分子的多肽和氨基酸,氨基酸浓度为5.23%,游离甲醛含量为37.34 mg/kg,重金属含量低于氨基酸肥料标准;将酶解液培养小麦种子后,能够促进小麦提前1 d生根,培养6 d的植株鲜重要优于对照。用清洁产品处理制革固废,不仅避免二次污染,还可对固废进行资源化利用。     

六种蛋白酶酶解效果的研究

本实验利用六种蛋白酶(1、2、3、4、5、6号蛋白酶)分别对豆粕、玉米芽粕、花生粕、菜籽粕、酒糟、黄粉虫、地鳖虫进行酶解,以肽含量为指标研究六种蛋白酶的酶解效果.结果显示,3号蛋白酶对酒糟、黄粉虫、地鳖虫酶解效果明显,酶解液肽含量最高可达12.6 mg·L-1、17.66 mg· L-1和36 mg·L-1;4号蛋白酶对豆粕酶解效果明显,酶解豆粕肽含量达到17.47 mg·L一1;5号蛋白酶对花生粕酶解效果明显,酶解花生粕肽含量最高可达18.45 mg·L1;六种蛋白酶均能酶解菜籽粕,酶解液肽含量均在15 mg·L-1以上,且差异不大.     

不同酶对草鱼蛋白功能特性及风味成分的影响

通过蛋白酶对草鱼进行酶解,对其酶解产物的功能特性和酶解液的风味成分进行研究。结果显示,风味蛋白酶、胰蛋白酶酶解产物的溶解性、热稳定性均优于胃蛋白酶酶解产物(P0.05)。经酶处理后鱼肉酶解液鲜甜味氨基酸占比增加,苦味氨基酸占比减少;利用SPME-GC-MS共检测出83种挥发性成分,以醛酮类和醇类为主,酶解处理后醇类的相对含量显著减少,醛酮类的相对含量则显著增加,酶解液具有独特风味。经胰蛋白酶、风味蛋白酶酶解后的草鱼肉酶解产物及酶解液均可作为食品基料应用于加工中。     

不同温湿条件下贮藏的3种禾本科植物花粉的一些生理变化

测定了水稻、玉米和狼尾草花粉在低温(4℃),低湿(RH45％),低温(4℃)高湿(RH90％),高温(35℃),低湿(RH45％)和高温(35℃),高湿(RH90％)4种贮藏条件下的蛋白酶活性,蛋白质含量,游离氨基酸含量的变化,结果表明：在低温高湿条件下,蛋白质降解和游离氨基酸含量上升慢;在高温条件下蛋白质降解快,游离氨基酸含量迅速上升。3种植物花粉中,水稻花粉蛋白酶活性强,在贮藏过程中蛋白质降解速率和游离氨基酸含量上升快：狼尾草花粉蛋白酶活性低,在贮藏过程中蛋白质降解速率和游离氨基酸含量上升慢;玉米花粉的蛋白酶活性、蛋白质降解速率和游离氨基酸含量上升速率居两者之间。     

竹叶青蛇毒丝氨酸蛋白酶的分子克隆和序列比较

利用逆转录酶与聚合酶链反应相结合的RT—PCR法,扩增出5个竹叶青(Trimeresurus stejnegeri)蛇毒丝氨酸蛋白酶的cDNAs;将扩增的cDNA片段克隆入pGEM-T载体中,筛选得到它们的基因,分别命名为TSSP-1、TSSP-2、TSSP-3、TSSP-4和TSSP-5。经末端终止法测定核苷酸序列,推导出5个丝氨酸蛋白酶的全序列;结合纯化的蛋白酶N-末端序列测定结果,推导TSSP-2、-3和-4分别编码凝血酶样酶stejnobin、纤溶酶stejnefibrase 1和2。5个丝氨酸蛋白酶分别含有1～6个N-型糖基结合位点,表明它们的计算分子量与纯化蛋白表观分子量之间的差异是由糖含量的不同造成,而其氨基酸序列相似度在60％～90％。TSSP-1和-2编码的成熟蛋白酶由236个氨基酸残基组成,TSSP-3、-4和-5的则由234个氨基酸残基组成。TSSP-1编码的蛋白酶在组成丝氨酸蛋白酶三联体催化活性中心产生了His^41-Arg^41的天然突变,这与其他自然界已发现的丝氨酸蛋白酶明显不同。     

角蛋白酶研究进展

角蛋白酶（keratinase）是分解角蛋白的一类蛋白酶,应用广泛,如分解含角蛋白的毛发、羽毛、角蹄、鳞等等,而角蛋白的化学结构稳定,不溶于水,一般的蛋白酶对它们降解不起什么作用。只有角蛋白酶对角蛋白有分解活性,所以称这种酶为角蛋白酶。     

中国长白山乌苏里蝮蛇金属蛋白酶解整合蛋白Ussurin基因克隆和序列分析

采用Clontech链转换建库试剂盒 ,建立了中国长白山乌苏里蝮蛇毒腺cDNA文库 ,从中克隆了金属蛋白酶 解整合蛋白Ussurin ,并进行了序列分析。结果显示 ,Ussurin开框读码序列由 14 34bp组成 ,编码 4 78个氨基酸。由核苷酸顺序推导的氨基酸序列可以看出 ,Ussurin最初的翻译产物是酶原前体 ;依次含有 18氨基酸组成的信号肽 ,171氨基酸组成的酶原区和由 2 89氨基酸组成的Ussurin(2 0 0氨基酸组成的金属蛋白酶结构域、16氨基酸组成的间隔区和 73氨基酸组成的解整合蛋白结构域 )。Ussurin的金属蛋白酶结构域含有 3对二硫键 ;解整合蛋白结构域含有 6对二硫键和特征性RGD(精氨酸 甘氨酸 天冬氨酸 )结构。其基因序列和结构域组成与GenBank中蛇毒金属蛋白酶 解整合蛋白呈现高度同源性属于P Ⅱ。氨基酸序列blast比对发现 ,酶原区和解整链蛋白结构域呈现极高的同源性 ,而金属蛋白酶结构域却出现了极高的变异 ,推测这些变异结构区是为了适应不同的底物、不同受体或同一受体的不同结构域     

鱼腥草游离氨基酸组成及含量的HPLC分析

研究不同鱼腥草材料间及其两个不同采收时期游离氨基酸组成及含量差异 ,筛选游离氨基酸种类较多 ,含量较高的新品系 ,并确定适宜的采收时期。对 1 9份鱼腥草材料阴干地上部分游离氨基酸成分进行了高效液相色谱分析。 6月和 1 0月采收的鱼腥草中分别含有 1 5种和 1 4种氨基酸 ,都不含Cysteine -ss -Cysteine和Lysine,1 0月采收的还不含有Tyrosine。氨基酸中Proline含量最高 ,Glycine含量最低。此外 ,1 0月采收的鱼腥草的游离氨基酸总量高于 6月采收的鱼腥草。供试材料中 6月采收的W0 1 - 86和 1 0月采收的W0 1 - 5多数游离氨基酸含量均为同一采收时期的最高值。分别为 5 894 .76mg/kg和 6 1 6 6 .1 3mg/kg。此外 ,蕺菜与峨眉蕺菜间游离氨基酸成分间无显著差别。因此鱼腥草不同材料间及不同采收时期游离氨基酸含量不尽相同 ,游离氨基酸含量与染色体数目间相关不显著     

Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops

Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from the thermophile Bacillus sp. WF146, revealed that the WF146 protease was translated as a 416-amino acid precursor consisting of a putative 18-amino acid signal peptide, a 10-kDa N-terminal propeptide and a 32-kDa mature protease region. The mature WF146 protease shares a high degree of amino acid sequence identity with two psychrophilic subtilisins, S41 (68.2%) and S39 (65.4%), and a mesophilic subtilisin, SSII (67.1%). Significantly, these closely related proteases adapted to different temperatures all had four inserted surface loops not found in other subtilisins. However, unlike those of S41, S39 and SSII, the inserted loops of the WF146 protease possessed stabilizing features, such as the introduction of Pro residues into the loop regions. Interestingly, the WF146 protease contained five of the seven mutations previously found in a hyperstable variant of subtilisin S41 obtained by directed evolution. The proform of WF146 protease (pro-WF146 protease) was overexpressed in Escherichia coli in an inactive soluble form. After heat treatment, the 42-kDa pro-WF146 protease converted to a 32-kDa active mature form by processing the N-terminal propeptide. The purified mature WF146 protease hydrolyzed casein with an optimum temperature of 85 degrees C, and lost activity with a half-life of 30 min at 80 degrees C in the presence of 10 mM CaCl2.     

鹅新城疫病毒P基因的RNA编辑及其相关蛋白的分子特征

应用RT-PCR方法对鹅新城疫病毒安徽分离株WF00GP基因进行了RNA编辑分析,发现w‰GP基因在保守的编辑位点上插入一个G,使得P基因增加编码V蛋白,而且它的最大ORF的长度为1188bp,编码395个氨基酸的P蛋白。wkG株和参考毒株的P基因的同源性和系统发育分析表明,WF00G株与水禽源毒株NA．1、ZJ1、FPI／02、PX2／03、Duck／1／05和SF02等亲缘关系较近,与传统疫苗株或弱毒株HB92、LaSota和Clone30以及F48E9等的亲缘关系相对较远。进一步对其V蛋白羧基端序列分析发现,虽然编辑位点氨基酸序列（132KKG134）和羧基端的5个Cys残基位点是高度保守的,但是V蛋白c末端氨基酸存在较大变异,APMV-1毒株V蛋白羧基端氨基酸的突变呈现“基因型一致性”。     

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.     

高温蛋白酶制备酶解猪血蛋白的研究

用高温蛋白酶ＷＦ１４６制备猪血水解蛋白得率在１０％（Ｗ／Ｖ）以上。水解液经分析含有１８种氨基酸,其中８种必需氨基酸占３７．１４％。氨基酸总量可达６２６４．８６ｍｇ／１００ｍｌ．产物具有较高的营养价值和良好的溶解性,因而在食品工业中具有很大的应用潜力。     

Cold-adapted maturation of thermophilic WF146 protease by mimicking the propeptide binding interactions of psychrophilic subtilisin S41

Thermophilic WF146 protease matures efficiently at 60 degrees C, but quite slowly at low temperatures. In this report, seven amino acid residues involved in interactions between the mature domain and the propeptide of the enzyme were substituted by corresponding residues of psychrophilic subtilisin S41 to generate mutant Mut7 (S105G/G107D/Y117E/S136N/V143G/K144E/D145S). Mut3 (S105G/G107D/Y117E) and Mut4 (S136N/V143G/K144E/D145S) were also constructed. Transferring structural features from S41 endowed Mut7 with a remarkably increased maturation rate, as well as an improved caseinolytic activity at 25 degrees C. Moreover, Mut3 and Mut4 each obtained one of the above endowments. Further studies suggest that low-temperature activity and maturation rate are not necessarily linked, and uncoupling structural elements modulating the two properties may be advantageous to cold adaptation.     

抗植物病毒农药“病毒煞”的氨基酸成分分析

本文通过对抗植物病毒农药“病毒煞”的氨基酸成分进行分析,共含有１８种氨基酸,其中脯氨酸、谷氨酸、精氨酸和天冬氨酸含量较高,约占水解氨基酸总量的４７％;含有１９种游离氨基酸,脯氨酸含量最高,占游离氨基酸总量的５１％,说明氨基酸可能是其防病增产的有效成分之一。     

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

The incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The half-life of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (T_m) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of K_m and k_cat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications.     

Process development for the enzymatic hydrolysis of food protein: Effects of pre-treatment and post-treatments on degree of hydrolysis and other product characteristics

An enzymatic process was developed to produce protein hydrolysate from defatted soya protein. Various unit operations were tried, and the effects of pre- and post-treatments on the product characteristics such as degree of hydrolysis (DH), free amino acid content (%FAA) and average molecular weight (MW) were investigated. The use of acid washes showed no difference in %DH. Increasing pH during pre-cooking gave lower %DH. Alkaline cooking made too much insoluble protein, thus the protein yield was too small. A better hydrolysis with more acceptable taste was obtained when the combination of Neutrase/Alcalase/Flavourzyme was used in place of Alcalase/Flavourzyme combination. Untoasted defatted soya was more effective on the proteolysis than toasted one. The MW of the evaporated and spray dried product was higher than that of undried product, due to precipitation of low-solubility components. When the product separation was carried out by ultrafiltration and the product concentration by reverse osmosis, the solubility and the taste of the product were improved. The difference between enzyme hydrolysate and acid hydrolysate was significant in free amino acid composition, especially in tyrosine, phenylalanine, glutamine and asparagine.     

A Protein Hydrolysate Enzymatically Produced from the Industrial Waste of Processing Icelandic Scallop Chlamys islandica

Dry hydrolysate was prepared from protein-containing waste of Icelandic scallop processing by means of a proteinase complex from king crab hepatopancreas. The resulting product contains no less than 80% free amino acids and oligopeptides. Predominant are aspartic acid, leucine, isoleucine, arginine, and lysine, which account for more than 50% of the free amino acids. The potential of using the protein hydrolysate as a nutrient for microorganism cultivation is estimated. It is demonstrated that the hydrolysate can be used for growing test cultures.     

Comparative kinetics of appearance in the portal vein of alpha-aminated nitrogen from mixtures of small peptides of free amino acids of the same composition introduced in the duodenum of conscious pigs

The quantitative kinetics of appearance of amino acids (a.a.) in the pig portal vein was studied in 6 animals for 5 hrs. after duodenal perfusion of an enzymatic hydrolysate of milk proteins or a solution of free a.a. of the same composition. Each product was given in two quantities (55 and 110 g). The quantities of a.a. appearing in the portal vein were higher after perfusion of the hydrolysate than after that of the free a.a., independently of the time after the perfusion. Thus, nitrogen present in the small intestine as small peptides is absorbed more quickly than when it is present as free amino acids.