蚕蛹蛋白质的酶—酸两步水解

将木瓜蛋白酶催化水解和酸水解联合起来应用于水解蚕蛹蛋白质制备氨基酸。与单纯酶解(水解时间48h)或酸水解(水解时间24h)相比,水解时间分别缩短了2/3和1/3,后处理简化,能耗降低。研究了蚕蛹蛋白质水解的影响因素,在适宜条件下,得到有香味的混合氨基酸,收率为98.5％(对蛋白质),其中含有17种游离氨基酸(色氨酸未测),含量为47.9％。

蚕蛹蛋白水解液的脱色条件研究

采用酶—酸两步水解法制备蚕蛹蛋白复合氨基酸。比较不同吸附剂对水解液的脱色效果,选择粉末活性炭作为蚕蛹蛋白水解液的脱色吸附剂。探讨吸附剂用量、脱色时间、脱色温度、水解液pH值4个因素对水解液脱色率和氨基酸损失率的影响,采用L9(34)正交试验设计对脱色条件进行优化,得到最优脱色条件为:吸附剂用量0.20g/(50mL1%蛋白提取液),脱色温度30℃,pH值5.0,脱色时间15min。在优化试验条件下,蚕蛹蛋白水解液平均脱色率达到91.27%,氨基酸损失率为9.12%。

鸭蛋清提取天然氨基酸的创新性和可行性

鸭蛋清含有丰富的氨基酸，是提取天然氨基酸的一个新颖途经。在氨基酸生产的主要方法中，其中酶一酸联合水解法是目前生产氨基酸方法中较好的一种，所以利用酶-酸联合水解鸭蛋清提取氨基酸，具有可行性。同时利用双酶-酸酸水解鸭蛋清提取氨基酸的研究还未见报道，具有创新性，也显示其广泛的应用前景。

Antioxidant Activity of Peptides Obtained from Cotton Ground Oil-Cake Proteins

Antioxidant activity of the peptides derived from proteins of defatted cottonseed kernels and cotton ground oil-cake by their enzymatic hydrolysis with acidic （Asp. niger） and neutral proteinases （Bac. amyloliquefaciens） was studied. Antioxidant activity of the derived peptides depended on the used proteins and enzymes. The peptides derived by using of neutral proteinase possessed higher antioxidant activity, in comparison with the peptides derived by acidic proteinases.

Immobilization of Aspergillus Oryzae Mycelium Pellets and Its Application in the Resolution of D, L-Alanine

Aspergillus oryzae 3042 mycelium pellets were immobilized by crosslinking method with reagents of gelatin and formaldehyde. An orthogonal design table was used to determine the optimal immobilization conditions. The L-aminoacylase activity of immobilized mycelium pellets under optimal conditions was assayed. The results show that the L-aminoacylase activity was 1350 U·g^-1 and the activity retention rate was 83%. The properties of the immobilized preparation were studied. Compared with free pellets, the appropriate pH of reaction system changed from 7.0 to 8.0, temperature changed from 52℃ to 63℃, and the ranges of the optimum reaction conditions were all improved. The effect of Co^2＋ on immobilized mycelium pellets was investigated and the favorable concentration was determined. When the immobilized preparation was used for the resolution of D, L-alanine in a packed bed reactor with 10 mm in diameter, 200 mm in height, the operational stability was increased, and the half-life period was 53 d. Thermal stability analysis shows that the immobilized pellets were more stable than the free pellets.

Nutritional evaluation of caseins and whey proteins and their hydrolysates from Protamex

Whey protein concentrate (WPC 80) and sodium caseinate were hydrolyzed by Protamex to 5%, 10%, 15%, and 20% degree of hydrolysis (DH). WPC 80, sodium caseinate and their hydrolysates were then analyzed, compared and evaluated for their nutritional qualities. Their chemical composition, protein solubility, amino acid composition, essential amino acid index (EAA index), biological value (BV), nutritional index (NI), chemical score, enzymic protein efficiency ratio (E-PER) and in vitro protein digestibility (IVPD) were determined. The results indicated that the enzymatic hydrolysis of WPC 80 and sodium caseinate by Protamex improved the solubility and IVPD of their hydrolysates. WPC 80, sodium caseinate and their hydrolysates were high-quality proteins and had a surplus of essential amino acids compared with the FAO/WHO/UNU (1985) reference standard. The nutritive value of WPC 80 and its hydrolysates was superior to that of sodium caseinate and its hydrolysates as indicated by some nutritional parameters such as the amino acid composition, chemical score, EAA index and predicted BV. However, the E-PER was lower for the WPC hydrolysates as compared to unhydrolyzed WPC 80 but sodium caseinate and its hydrolysates did not differ significantly. The nutritional qualities of WPC 80, sodium caseinate and their hydrolysates were good and make them appropriate for food formulations or as nutritional supplements.

DETERMINATION OF AMINO ACIDS IN TWO POLYSIPHONIA SPECIES AND STUDY OF ENZYMATIC HYDROLYSIS METHOD

The total content of the rich amino acids in two common red algae, Polysiphonia urceolata and Polysiphonia japonica growing in the Qingdao seashore were determined. The algae powder was hydrolyzed by 6 mol/L HCl at 110℃ for 48 h and determined by amino acid analyzer. The content was 25.35% and 24.16%, respectively, much higher than that of some other species. In addition, a nutritive liquid with abundant amino acids was prepared (by the enzymatic hydrolysis method using Polysiphonia urceolata ) as raw material for a kind of health beverage. The dried seaweed was decolored by 0.25% KMnO 4 and 0.5% active carbon, then enzymalized. In the selection of enzymalizing condition, the orthogonal experimental design was used. Four factors including kinds of enzyme, quantity of enzyme, temperature and time were studied at 3 levels. According to the orthogonal design results, we can choose an optimal condition: hydrolyzing at 45℃ by neutral proteinase (0.25%, w/w) for 2 h, adjusting pH to 8.5, then adding trypsin (0.25%, w/w) and hydrolyzing for 2 h. Finally the above solution was alkalized by NaOH and neutralized by casein. After the hydrolyzed liquid was filtered and concentrated, suitable additives were added. The final products contain rich amino acids.

Purification and characterization of an arginine ester hydrolase from the venom of Trimeresurus mucrosqumatus in Hunan province of China

Objective: To study the physical and chemical properties of an arginine ester hydrolase from the venom of Trimeresurus mucrosqumatus in Hunan province of China.Methods:The arginine ester hydrolase(AEH) was isolated from the venom of Chinese Trimeresurus mucrosqumatus by a combination of ion-exchange chromatography on DEAE-Sephadex A-50, CM-Sepharose Cl-6B and gel filtration on Sephadex G-100.Results: The purified protein named TM-AEH,a glycoprotein with carbohydrate content of 0.5% neutral hexose and 0.75% sialic acid,a relative molecular mass of 29.0 kDa,and an isoelectric point(pI) of 5.2. It shares with an extinction coefficient(E 0.1%/cm) of 1.332 at 280 nm,consisted of 225 amino acid residues,and migrated as a band under reduced or non-reduced condition in basic PAGE.TM-AEH was a highly thermostable protein and was stable to pH changes between 5 and 9.The optimum temperature and optimum pH were 55℃ and 8.4 for its catalytic activity respectively,which was inhibited by Fe 3+ and Cu 2+.Conclusion:This protein can exhibit higher BAEE-hydrolysing activity and fibrinogenolytic activity as compared to that of whole venom.

Separated Hydrolysis and Fermentation of Water Hyacinth Leaves for Ethanol Production

Water hyacinth is a raw material for long-term sustainable production of cellulosics ethanol. In this study, the acid pretreatment and enzymatic hydrolysis were used to evaluate to produce more sugar, to be fermented to ethanol. Separated hydrolysis and fermentation （SHF） studies were carried out to produce ethanol from water hyacinth leaves. Dilute sulfuric acid pretreatment and enzymatic hydrolysis were conducted to select the optimum pretreatment conditions. The optimum pretreatment conditions included T = 135 ℃, t = 30 min, and sulfuric acid concentration = 0.1 M. The residue was enzymatically hydrolyzed using the mixture of enzymes cellulase, xylanase and pectinase. The maximum enzymatic saccharification of cellulosic material （76.8%） was achieved. SHF by mono-culture of Saccharomyces cerevisiae KM1195 achieved the highest yields of ethanol. Furthermore, ethanol production was accomplished with the co-culture ofS. cerevisiae TISTR5048 and Candida tropicalis TISTR5045 which produced the highest increase in ethanol Yield. In this case, the ethanol concentration of 3.42 （g/L）, percentage of the theoretical ethanol yield of 99.9%, the ethanol yield of 0.27 g/g and the productivity of 0.22 g/L/h were obtained. This suggested that mild acid pretreatment and co-cultureare promising methods to improve enzymatic hydrolysis and ethanol production from water hyacinth.

Adeno-associated virus vector-mediated triple gene transfer of dopamine synthetic enzymes

Objective To explore triple gene transfer of dopamine synthetic enzymes with separate adeno-associated virus (AAV) vectors.Methods The genes for dopamine synthetic enzymes, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and GTP cyclohydrolase Ⅰ (GCH, an enzyme critical for tetrahydrobiopterin synthesis) were cotransduced into 293 cells with separate AAV vectors. Expressions of TH, AADC and GCH were detected by Western blot analysis. Intracellular dopamine level was assayed by high-performance liquid chromatography.Results TH, AADC and GCH were effectively coexpressed in transduced cells with three separate AAV vectors, AAV-TH, AAV-AADC and AAV-GCH. Furthermore, the coexpression resulted in an effectively spontaneous dopamine production in cotransduced cells.Conclusion The triple transduction of TH, AADC and GCH genes with separate AAV vectors is effective, which might be important to gene therapy for Parkinson's disease.

Cloning and characterization of an oxiranedicarboxylate hydrolase from Labrys sp. WH-1

Objective:This study aimed to clone and characterize the oxiranedicarboxylate hydrolase(ORCH) from Labrys sp.WH-1.Methods:Purification by column chromatography,characterization of enzymatic properties,gene cloning by protein terminal sequencing and polymerase chain reaction(PCR),and sequence analysis by secondary structure prediction and multiple sequence alignment were performed.Results:The ORCH from Labrys sp.WH-1 was purified 26-fold with a yield of 12.7%.It is a monomer with an isoelectric point(pl) of 8.57 and molecular mass of 30.2 kDa.It was stable up to 55℃with temperature at which the activity of the enzyme decreased by 50% in 15 min(T5015) of 61℃and the half-life at 50℃(t1/2,50℃) of 51 min and was also stable from pH 4 to 10,with maximum activity at 55℃and pH 8.5.It is a metal-independent enzyme and strongly inhibited by Cu2+,Ag+,and anionic surfactants.Its kinetic parameters(Km,kcat,and kcat/Km) were 18.7 mmol/L,222.3 s-1,and 11.9 mmol/(L s),respectively.The ORCH gene,which contained an open reading frame(ORF) of 825 bp encoding 274 amino acid residues,was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain.Conclusions The catalytic efficiency and thermal stability of the ORCH from Labrys sp.WH-1 were the best among the reported ORCHs,and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.