鸭毛水解制备复合氨基酸新工艺的研究

采用正交试验方法,对鸭毛水解制备复合氨基酸工艺条件进行优选,试验结果表明,鸭毛水解的最佳工艺条件为：水解时间10小时,反应温度100℃,盐酸浓度为4摩尔/升。     

从鸡毛中提取复合氨基酸的研究

运用正交实验研究了以鸡毛为原料硫酸水解生产复合氨基酸的水解条件。结果表明 :硫酸浓度为 8mol·L- 1 ,鸡毛质量 (g)与硫酸体积 (mL)之比为 1∶2 .5 ,水解时间 10h ,其水解率可达 5 5 .2 3%。得到的固体氨基酸质量分数为 95 .31%。     

猪血粉制备复合氨基酸工艺研究

本文采用正交试验法优选硫酸水解猪血粉制备复合氨基酸的最佳条件,经中试及工业规模生产证明:复合氨基酸收率平均为53.3％,氨基酸含量为72.7％。     

由铬革屑制备可溶性蛋白及氨基酸的研究

成品铬革屑中蛋白质含量在 70 %以上 ,处理成小颗粒后与水、Ca(OH) 2 按 1∶4 0∶0 .4的比例混合 ,90℃反应 5h ,可溶性蛋白转化率近 6 0 % ,且在升温后加入Ca(OH) 2 有利于退鞣。以 6mol/L盐酸水解皮革蛋白粉制备复合氨基酸 ,水解 14h ,水解液以HD -I树脂脱色 ,氨基酸损失较少 ,且动态脱色效率明显高于静态脱色 ,采用 717树脂脱酸可获得pH4 .5 - 5的复合氨基酸溶液 ,总得率为 6 0 .1%。     

盐酸催化水解槐角异黄酮的研究

利用盐酸甲醇溶液对槐角总异黄酮进行水解制备染料木黄酮。采用高效液相色谱法检测槐角异黄酮水解率。探讨水解时间、盐酸浓度和水解温度对异黄酮水解率的影响,并通过响应面法确定最佳水解条件。实验结果表明最佳水解条件为:水解时间3.8 h、盐酸浓度2.59 mol/L、水解温度78.5℃。在此工艺下,槐角异黄酮水解率达93.38%。     

芦苇浆纳米纤维素超声法制备工艺优化及表征

以芦苇浆为原料,采用超声辅助硫酸水解法制备了纳米纤维素.在单因素实验的基础上,响应面法优化纳米纤维素制备工艺条件,结果表明最佳制备工艺条件为超声时间32 min,硫酸浓度52％,反应温度54℃,纳米纤维素得率最高(78.67％);通过傅里叶变换红外(FTIR)、X射线衍射和透射电子显微镜(TEM)对最佳工艺条件制备的纳米纤维素进行性能表征,分析表明最佳工艺条件制备的纳米纤维素聚集态结构为纤维素Ⅰ型,呈棒状.     

二次中和法制取菜籽复合氨基酸新工艺研究

为得到高质量的菜籽复合氨基酸,以脱皮菜籽粕为原料,研究了硫酸水解制备复合氨基酸的新工艺一二次中和法,解决了氨基酸脱色和副产品植酸的利用问题。水解的最佳工艺参数是3mol·L-1的H2SO4水解18h;ca(OH)2作中和剂,第一次中和和脱色的最佳pH为2-4,第二次中和的最佳pH为6.5-7.0,中和后的溶液经减压浓缩,喷雾干燥,制得菜籽复合氨基酸。复合氨基酸得率≥40％,纯度≥40％;副产品植酸钙得率10％左右,纯度40％-50％。     

杜仲叶黄酮苷元水解及富集纯化工艺研究

以杜仲叶提取物为原料,采用酸水解的方法制备杜仲叶黄酮苷元,通过单因素及正交试验优化杜仲叶黄酮苷元的水解条件;并研究了聚酰胺两步法富集黄酮苷元的工艺条件。结果表明:当料液比为2∶1(mg/m L)时,杜仲叶黄酮苷元水解的最佳工艺参数为:水解温度80℃,水解时间3 h,盐酸浓度6 mol/L,黄酮苷元的含量可达到2.512%。水解样品经过一次聚酰胺静态吸附,黄酮苷元的纯度可达12.87%,再经聚酰胺二次富集,杜仲黄酮苷元纯度可达63.19%。     

水解乳蛋白的制备及在细胞培养中的初步应用

采用正交法优化复合酶(胰酶和中性蛋白酶)水解凝乳酶干酪素制备水解乳蛋白(lacto-protein hydrolysate,LPH)的制备工艺,通过BHK和Vero细胞的培养效果检测水解乳蛋白的促细胞生长作用。结果显示水解乳蛋白的最佳制备工艺为:胰酶∶中性蛋白酶=2∶1(质量比),40℃,pH 7.0,酶/底物(E/S)=1∶5(质量比)水解6 h。所得产物氨基氮含量3.21 g/L,多肽含量35.63 g/L,游离氨基酸含量14.17 mg/mL,收率达40.20%。BHK和Vero细胞培养24 h,细胞密度均为各自空白的2.0倍,分别为Hyclone产品的1.0倍和0.86倍;培养48h,分别为各自空白的5.0倍和4.0倍,均为Hyclone产品的0.88倍。因此,该工艺简单,耗时短,制备得水解乳蛋白对BHK和Vero细胞生长有明显的促进作用。     

鲜猪血加压水解制备复合氨基酸新工艺的研究

采用正交试验方法,对鲜猪血加压水解制取复合氨基酸工艺条件进行优选。试验结果表明,采用鲜猪血加压水解制取复合氨基酸与采用猪血粉常压水解相比,简化了生产工序,缩短水解时间８～１２小时,降低能耗２倍多,减少硫酸用量２～３倍,而产品质量稳定可靠。     

Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase.

We report the recombinant neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05-0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025-0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (DeltaH*) and entropy (DeltaS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.     

Capacity of aspartic acid to increase the bacterial count on suspensions of Escherichia coli after freezing

The addition of 2% Trypticase to a minimal salts-glucose plating medium increased the bacterial count of frozen and thawed suspensions of Escherichia coli 451B cells, even when precautions were taken to remove toxic trace elements from the plating diluent. Hydrolysis of the Trypticase with HCl or H(2)SO(4) reduced its count-increasing activity. Treatment of the H(2)SO(4) hydrolysate with a cation-exchange resin greatly improved its capacity to replace Trypticase. Addition of a mixture of amino acids approximating the composition of casein also increased the plate count when added at a level equivalent to 0.1% casein, but at 2% it depressed the count. Tests of amino acids in the mixture revealed that aspartic acid could replace Trypticase completely as a supplement to the basal medium. When added at a level of 2.5 mm, aspartic acid doubled and occasionally tripled the plate count of a suspension of frozen and thawed cells. Glutamic acid, alanine, and to a lesser extent certain other amino acids also showed a capacity to increase the count. Cysteine was without significant effect. Serine and other amino acids depressed the count. None of the amino acids or other supplements affected the count of suspensions of cells that had not been frozen. The effect of adding aspartic acid, cysteine, or Trypticase to the basal medium on the bacterial count of suspensions of various strains of E. coli, Aerobacter aerogenes, Serratia marcescens, and two species of Pseudomonas after freezing was examined. The response to the supplements was unique for each organism.     

Characteristics of chymotrypsin modified with water-soluble acylating reagents and its peptide synthesis ability in aqueous organic media.

Several kinds of modified chymotrypsin were prepared with water-soluble acylating reagents, and their characteristics after hydrolyzing with unmodified chymotrypsin in aqueous-N,N'-dimethylformamide (DMF) media were compared. It was found that chymotrypsin (Csin), of which a 20% amino group was modified with a benzyloxycarbonyl group (Z(20)Csin), had more favorable characteristics than unmodified chymotrypsin with regard to hydrolytic activity in an aqueous DMF media. We also investigated the Z(20)Csin-catalyzed peptide synthesis in two different solution systems. In the one-layer system containing water and DMF, Z(20)Csin catalyzed the peptide bond formation in a higher yield than that by unmodifide chymotrypsin and enabled a synthetic reaction in even an 80% (v/v) DMF media, in which the hydrolytic reaction could not be carried out. Z(20)Csin catalyzed the condensation between some N-acyl amino acids or peptide derivatives and amino acids in 90% ethylacetate, 90% hexane or 50% benzene. This latter method employs a two-layer system, and the modified enzyme may be able to reduce the number of synthetic steps when preparing acyl peptides.     

Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.     

Structures of Gibberellins A33 and A34 from Immature Seeds of Calonyction aculeatum

Several kinds of modified chymotrypsin were prepared with water-soluble acylating reagents, and their characteristics after hydrolyzing with unmodified chymotrypsin in aqueous-N,N’ -dimethylformamide (DMF) media were compared. It was found that chymotrypsin (Csin), of which a 20% amino group was modified with a benzyloxycarbonyl group (Z(20)Csin), had more favorable characteristics than unmodified chymotrypsin with regard to hydrolytic activity in an aqueous DMF media. We also investigated the Z(20)Csin-catalyzed peptide synthesis in two different solution systems. In the one-layer system containing water and DMF, Z(20)Csin catalyzed the peptide bond formation in a higher yield than that by unmodifide chymotrypsin and enabled a synthetic reaction in even an 80% (v/v) DMF media, in which the hydrolytic reaction could not be carried out. Z(20)Csin catalyzed the condensation between some N-acyl amino acids or peptide derivatives and amino acids in 90% ethylacetate, 90% hexane or 50% benzene. This latter method employs a two-layer system, and the modified enzyme may be able to reduce the number of synthetic steps when preparing acyl peptides.     

Prebiotic synthesis in atmospheres containing CH4, CO,and CO2

The prebiotic synthesis of organic compounds using a spark discharge on various simulated primitive earth atmospheres at 25 degrees C has been studied. Methane mixtures contained H2 + CH4 + H2O + N2 + NH3 with H2/CH4 molar ratios from 0 to 4 and pNH3 = 0.1 torr. A similar set of experiments without added NH3 was performed. The yields of amino acids (1.2 to 4.7% based on the carbon) are approximately independent of the H2/CH4 ratio and whether NH3 was present, and a wide variety of amino acids are obtained. Mixtures of H2 + CO + H2O + N2 and H2 + CO2 + H2O + N2, with and without added NH3, all gave about 2% yields of amino acids at H2/CO and H2/CO2 ratios of 2 to 4. For a H2/CO2 ratio of 0, the yield of amino acids is extremely low (10(-3)%). Glycine is almost the only amino acid produced from CO and CO2 model atmospheres. These results show that the maximum yield is about the same for the three carbon sources at high H2/carbon ratios, but that CH4 is superior at low H2/carbon ratios. In addition, CH4 gives a much greater variety of amino acids than either CO or CO2. If it is assumed that an abundance of amino acids more complex than glycine was required for the origin of life, then these results indicate the requirement for CH4 in the primitive atmosphere.     

Specific sulfonation of tyrosine, tryptophan and hydroxy-amino acids in peptides

1. ClSO3H in trifluoroacetic acid rapidly converts serine and threonine into O-sulfate ester derivatives while tyrosine and tryptophan are converted into arylsulfonic acids. 2. H2SO4 in trifluoroacetic acid reacts more slowly with serine, threonine and tyrosine while is not able to modify tryptophan. 3. All other amino acids are perfectly stable under the above reaction conditions. 4. Peptides containing susceptible amino acid residues are specifically converted into the corresponding sulfonated derivatives in high or quantitative yield.     

An Improved Bioprocess for Extracellular l-Leucine Amino Peptidase Production Using Streptomyces gedanensis

A bioprocess was developed for the production of L-leucine aminopeptidase under solid-state fermentation (SSF) by cultivating Streptomyces gedanensis in an inert support impregnated with a minimal medium. Response surface methodology of Box Behnken design was used to derive the optimum level of significant factors (3 ml inoculum (1.2 × 10(9) CFU/ml); 0.275% w/v (NH(4))(2)SO(4); 0.275% w/v MgSO(4)·7H(2)O and 0.55% w/v Tryptone) for maximum LAP production (489 IU/g PUF) as compared to the initial level of 176.3 ± 0.02 IU/g PUF. The high level of extracellular aminopeptidase yield achieved in this work showed the technical feasibility of LAP production under SSF using inert support and is the first report of this kind. The ability of Streptomyces amino peptidase to release particular N-terminal amino acids made them interesting for controlling the degree of hydrolysis and flavor development for a wide range of substrates in food like industries.     

Regeneration of amino acids from thiazolinones formed in the Edman degradation.

Conditions for regeneration of amino acids from their thiazolinone derivatives formed in the cleavage step of the Edman degradation procedure have been investigated. Highest yields of most amino acids including methionine were obtained simply by hydrolysis in 5.7n HCl containing 0.1% SnCl₂ for 4 hr at 150°C. Results from other methods of hydrolysis are provided for comparison. The hydrolytic yield of amino acids from the anilinothiazolinones determined in this study may be used to estimate the cleavage yield at each step of the Edman degradation.     

Salt dependence, kinetic properties and catalytic mechanism of N-formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri.

N-Formylmethanofuran(CHO-MFR):tetrahydromethanopterin(H4MPT) formyltransferase (formyltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100-fold to apparent homogeneity with a 54% yield. The monomeric enzyme had an apparent molecular mass of 35 kDa. The N-terminal amino acid sequence of the polypeptide was determined. The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity. The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4. The salts KCl, NaCl and NH4Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half-maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required. A detailed kinetic analysis revealed that phosphates and sulfates both affected the Vmax rather than the Km for CHO-MFR and H4MPT. At the optimal salt concentration and at 65 degrees C, the Vmax was 2700 U/mg (1 U = 1 mumol/min), the Km for CHO-MFR was 50 microM and the Km for H4MPT was 100 microM. At 90 degrees C, the temperature optimum of the enzyme, the Vmax was about 2.5-fold higher than at 65 degrees C. Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization. The efficiency of salts in protecting formyltransferase from heat inactivation at 90 degrees C decreased in the order K2HPO4 = (NH4)2SO4 > KCl = NH4Cl = NaCl > Na2SO4 > Na2HPO4. The catalytic mechanism of formyltransferase was determined to be of the ternary-complex type. The properties of the enzyme from M. kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus.