固定化腈水解酶的制备及酶学性能

研究磁性纳米载体的氨基化修饰及其固定化腈水解酶的条件,探讨固定化腈水解酶的酶学性能.研究结果表明载体固定腈水解酶的最佳工艺为:腈水解酶与载体配比15mg/g、戊二醛浓度3%、固定化温度30℃、固定化时间60min.固定化腈水解酶的热稳定性及p H稳定性都比游离酶有很大提高.

蔗果糖酶生产菌株的筛选及生产特性研究

对七种黑曲霉菌种的果糖苷转移酶、水解酶活力进行了检测。以β－呋喃果糖苷酶活力为指标，经紫外线、Ｃｏ６０照射诱变，筛选出生产潜力较大的菌株ＨＮＣＯ－８００８并初步研究了该菌株的生产特性。本文对发酵体系中几种与寡果糖生产有关的酶活力的区分方法进行了较深入探讨。

Optimization of Hydrolysis Conditions for Corn Gluten Meal

[Objective] To investigate the optimal enzyme for the hydrolysis of corn gluten meal and the optimal hydrolysis conditions for the enzyme. [Method] Nine kinds of enzymes were used to hydrolyze the corn gluten meal, using the formaldehyde titration method for the determination of hydrolysis degree, and orthogonal test was used to determine the optimal hydrolysis conditions for double enzymes hydrol- ysis of corn gluten meal. [Result] The optimal pretreatment condition for corn gluten meal is heating at 121 ~C for 30 min. The double enzyme hydrolysis for the pro- treated corn gluten meal using 2709 alkaline protease and flavourzyme showed that the degree of hydrolysis could reach 32.4% with enzyme addition amount of 4%, hy- drolysis time of 4 h at 45℃ and pH=7.0. [Conclusion] This study laid the foundation for the study on the preparation of bioactive peptides such as oligopeptide with high F value and antihypertensive peptides, further improving the corn intensive process- ing industrial chain.

Comparative Study on Hydrolase Activities in Tianzhu White Yak Milk and Gannan Yak Milk of Gansu

This study was conducted to compare the hydrolase activities in the milk of Tianzhu white yak and Gannan yak in Gansu. The results showed that the ACP activity in Tianzhu yak milk was significantly higher than that in Gannan yak milk （P〈0.05）; the AKP activity in Tianzhu yak milk was higher than that in Gannan yak milk, but not significant; and the AMC activity in Tianzhu yak milk was significantly lower than that in Gannan yak milk （P〈0.05）. There were no remarkable changes in the activities of the 3 hydrolases with the change of parity, i.e., the effects of parity on enzyme activities in milk were not significant. The comparative study on hydrolase activities in the milk of Tianzhu while yak and Gannan yak helps to understand the characteristics of the milk of the 2 yak breeds, and further provides certain reference basis for the development of yak milk.

Effect of Lysophosphatidylcholine on ATP and ρ-Nitrophenyl Phosphate Hydrolysis by the Plasma Membrane H^+-ATPase from Soybean Hypocotyls

The stimulatory effect of lysophosphatidylcholine (lyso_PC) on ATP and ρ_nitrophenyl phosphate (PNPP) hydrolysis by the plasma membrane H +_ATPase from soybean (Glycine max (L.) Merr.) hypocotyls was studied. Results showed that lyso_PC stimulated the hydrolysis of ATP; ATP hydrolysis was enhanced dramatically when lyso_PC was within 0-0.03%, and increased slightly when lyso_PC was higher than 0.03%. At the concentration of 0.03%, lyso_PC stimulated ATP hydrolysis by 80.5%. Kinetics analysis showed that V max increased from 0.46 μmol P i·mg -1 protein·min -1 to 0.87 μmol P i·mg -1 protein·min -1 while K m increased from 0.88 mmol/L to 1.15 mmol/L under lyso_PC treatment. The optimum pH of ATP hydrolysis was shifted from 6.5 to 7.0 . Moreover, it was found lyso_PC enhanced the inhibition of ATP hydrolysis by hydroxylamine. In the presence of 200 mmol/L hydroxylamine, ATP hydrolysis was inhibited by 74.4%, while it was inhibited by 84.4% when treated with lyso_PC. However, PNPP hydrolysis and the inhibitory effect of vanadate were not affected by lyso_PC. The above results indicated that the kinase domain might be an action site or regulatory region of the C_terminal autoinhibitory domain in the plant plasma membrane H +_ATPase.

Effect of chitosan on tobacco mosaic virus(TMV) accumulation, hydrolase activity, and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus(TMV) in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV(2 μg/mL) and chitosan(1 mg/mL) were lower in the early period of infection(3 days after inoculation), by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases(proteases, RNases) in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid(PTA)-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles(18 nm in diameter, 300 nm long), these suspensions contained abnormal(swollen, "thin" and "short") virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that "thin" virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.

A Novel Exponential Kinetic Model for Casein Tryptic Hydrolysis to Prepare Active Peptides

The kinetics of casein tryptic hydrolysis to prepare activepeptides was investigated. Taking into account the reaction mechanismincluding single substrate hydrolysis, irreversible enzymeinactivation, and substrate inhibition, a set of exponentialequations was established to characterize the enzymatic hydrolysiscurves. The verification was carried out by a series of experimentalresults and indicated that the average regressive error was less than5/100. According to the proposed kinetic model, the kinetic constantsand thermodynamic constants of the reaction system were alsocalculated.

A Novel Hydrolytic Product from Flesh of Mactra veneriformis and Its Bioactivities in Calcium Supplement

To prepare calcium-binding peptides, the flesh residue of Mactra Veneriformis was subjected to enzymatic hydrolysis. By comparing the capability of combining calcium of the hydrolyzates, pepsin was confirmed to be the most suitable enzyme for hydrolyzing the flesh residue to release calcium-binding peptides among the seven tested proteases. The pepsin hydrolyzate (PHM) was divided into three fractions according to the molecule weight of its composition, which ranged from 0.5 to 15 kDa. The low-molecule-weight fraction named PHM-3 had the highest capability in combining calcium. The peptides existing in the PHM-3 fraction consisted of higher contents of Glu, Ala and Leu, and could produce one type of calcium-peptide complex by powerfully chelating calcium ions. PHM-3 products could effectively increase calcium absorption and retention while they decreased the calcium excretion in animal tests. Additionally, symptoms caused by low calcium bioavailability in ovariectomized rats, such as bone mineral density reduction and mechanical strength loss could be significantly ameliorated by the hydrolytic products addition in diet.

Antioxidation Activities of Low-Molecular-Weight Gelatin Hydrolysate Isolated from the Sea Cucumber Stichopus japonicus

Gelatin extracted from the body wall of the sea cucumber （Stichopus japonicus） was hydrolyzed with flavourzyme. Low-molecular-weight gelatin hydrolysate （LMW-GH） of 700-- 1700 Da was produced using an ultrafiltration membrane bioreaetor system. Chemiluminescence analysis revealed that LMW-GH scavenges high free radicals in a concentration-dependent manner; IC50 value for superoxide and hydroxyl radicals was 442 and 285 μgmL-1, respectively. LMW-GH exhibited excellent inhibitory characteristics against melanin synthesis and tyrosinase activity in B16 cells. Furthermore, LMW-GH notably increased in- traeellular glutathione （GSH）, which in turn suppressed melanogenesis. LMW-GH performs antioxidation activity, holding the potential of being used as a valuable ingredient in function foods, cosmetics and pharmaceuticals or nutriceuticals.

Selection of Strains Producing Lipase with Transesterification Activity and Its Characterization

The interest for lipase production is due to the ability of this enzyme to catalyze some reactions, such as the transesterification. Although industrial biodiesel is produced chemically, there are several problems associated with this technology that can be prevented through the use of lipases. The present work aimed to select microorganisms with potential for production of lipase with transesterification activity. The lipase from Burkholderia cepacia was the one with the most promising results for this type of reaction, showing results of hydrolytic activity at 37 ℃and pH 8.0. The pH and volume of crude enzyme extract that showed favorable for synthesis ofbiodiesel is at about pH 6.0 and 3.75 mL, respectively, which represents approximately 42% of water in the system, ensuring the conversion of nearly 60% to biodiesel.

Preparation of Active Peptides from Corn and Soybean by Bi-enzyme Hydrolysis

[Objective] The aim was to study the optimal test condition for preparation of active peptides from corn and soybean by bi-enzyme hy- drolysis. [Metbod]Taking soybean peptide and corn peptide as materials, different concentrations of soy-corn protein isolate solution were pre- pared. Through pretreatment, and based on compound protease, flavor protease, neutral protease, papaya protease, pineapple hydrolysis, and by measuring the degree of hydrolysis activity and enzyme activity, tested enzyme was determined. Mixing two of those enzymes in certain proportion and by orthogonal test, the optimal condition of enzyme hydrolysis of a double was determined. [ Result] The optimal hydrolysis condition was 2% of substrate concentration, 1 ： 1of hydrolyze compound protease and flavor protease, and 8 h of hydrolysis time, 75.14% of DH. [ Condasien] The study provided a basis for the development and application of active peptides from corn and soybean by bi-enzyme hydrolysis.

糖添加量对广式腊肠脂质氧化稳定性及感官品质的影响研究

研究了不同糖添加量(3%、6%、9%、12%,m/m)对广式腊肠脂质水解酶活、脂质氧化稳定性及产品感官品质的影响规律。结果表明中性脂肪酶、酸性脂肪酶和磷脂酶活力在加工过程中呈下降趋势(p<0.05)。中性脂肪酶活力高于酸性脂肪酶和磷脂酶活力。糖添加量显著影响三种脂肪酶活力在加工过程中的变化,高糖添加组的三种脂肪酶活力略高于低糖添加组;糖添加量为3%时,烘烤结束后中性脂肪酶、酸性脂肪酶和磷脂酶活力分别下降77%、81%和96%,而添加量为12%时,其分别下降67%、65%和78%。烘烤结束后低糖添加组的过氧化值、羰基值和己醛含量较高,表明糖添加量的降低导致脂质氧化的加剧。感官分析表明糖添加量的降低致使咸味更加突出且出现一定的脂质哈败味,降低了广式腊肠产品的接受度。

Biocatalytic desymmetrization of 3-substituted glutaronitriles by nitrilases. A convenient chemoenzymatic access to optically active (S)-Pregabalin and (R)-Baclofen

Desymmetrization of prochiral 3-substituted glutaronitriles offers a new approach to access （S）-Pregabalin and （R）-Baclofen. A number of nitrilases from diverse sources were screened with 3-isobutylglutaronitriles （1a） or 3-（4＇-chlorophenyl）glutaronitriles （1b） as the substrate. Some nitrilases were found to catalyze the desymmetric hydrolysis of la and lb to form optically active 3-（cyanomethyl）-5-methylhexanoic acid （2a） and 3-（4＇-chlorophenyl）-4-cyanobutanoic acid （2b） with high enantiomeric excesse （ee）, respectively. This cannot be achieved using traditional chemical hydrolysis. Among them, AtNIT3 generated （R）-2b whereas BjNIT6402 and HsN1T produced the opposite （S）-enantiomer with high conversions and ee values. Not only the nitrilases showed different activities and stereoselectivities toward these 3-substituted glutaronitriles, the 3-substitueut of the substrates also exerted great effect on the enzyme activity and stereoselectivity. （S）-2a and （S）-2b were prepared with high yields and ee values using BjNIT6402 and HsNIT as the biocatalysts, respectively. A straightforward Curtius rearrangement of （S）-2a and （S）-2b, followed by the acidic hydrolysis, afforded （S）-Pregabalin and （R）-Baclofen. This offers a new platform methodology for the synthesis of optically active β-substituted T-amino acids of pharmaceutical importance.