固体发酵制备毛霉蛋白酶的研究

研究了毛霉M4产蛋白酶的若干影响因素,包括培养基的成分以及发酵条件等。结果表明,在麸皮与水的合适比例为1∶0.8;在基本培养基基础上添加一定浓度的碳源,氮源及无机盐有利于蛋白酶的合成;菌体较为合适的产酶条件为温度28℃,pH6.0,发酵时间36h。

提高洗净毛白度的加酶洗毛工艺

实验研究表明，残留于净毛表面的蛋白质污染物对洗净毛白度有显著影响．本文根据蛋白质污染物的化学特性，提出了加酶洗毛可有效地去除蛋白质污染物，提高洗净毛白度；同时还探讨了酶浓度、温度和助剂等对洗涤效果的影响．

嗜热毛壳菌纤维酶对肉鸡消化道酶活性影响的研究

试验旨在研究嗜热毛壳菌纤维酶对肉鸡消化道酶活性的影响。选用1日龄健康AA肉仔鸡120羽,随机分成4组,即对照组和试验1、2、3组,分别添加嗜热毛壳菌纤维酶0、0.05%、0.15%和0.25%。结果表明,试验2和3组十二指肠(P<0.05)和空肠胰蛋白酶、胃蛋白酶的活性(P<0.01)以及十二指肠、空肠和胰脏脂肪酶的活性(P<0.05或P<0.01)显著或极显著高于其他各组,对淀粉酶的活性无显著影响(P>0.05)。

加酶洗毛工艺的研究

本文提出碱性蛋白酶附加到第三洗槽,有利于发挥酶的洗涤作用,同时得出了加酶洗毛最佳工艺条件。

驴皮低温酶解脱毛方法及其对阿胶质量的影响

[目的]开发一种适合阿胶产业化生产的高效驴皮脱毛方法。[方法]低温条件下(4℃),选用1%碱性蛋白酶和1%中性蛋白酶对醒发的干驴皮开展酶解脱毛试验,比较2种酶解方法处理下驴皮的脱毛时间、脱毛率,并对脱毛过程中驴皮的气味进行评价。以1%碱性蛋白酶和1%中性蛋白酶酶解处理驴皮以及机械脱毛鲜驴皮为材料熬制阿胶,比较不同原料熬制阿胶的出胶量、出胶率、外观、气味等;测定以2种蛋白酶酶解处理的驴皮为原料熬制的阿胶质量指标,包括水分、灰分、水不溶物以及4种氨基酸含量,并与《中国药典》2015版一部中提供的相应质量标准进行对比。[结果]低温条件下(4℃),利用1%碱性蛋白酶(pH值=10.5)或1%中性蛋白酶(pH值=7.5)酶解处理驴皮第2天表现出脱毛效果,处理第5天醒发的干驴皮脱毛率可达90%,有臭味产生但非驴皮腐败臭味,而是脱毛过程中产生的正常气味。以1%碱性蛋白酶、1%中性蛋白酶酶解脱毛后的驴皮以及机械脱毛鲜驴皮为原料熬制阿胶,出胶量分别为113.0、109.4、129.3 g,出胶率分别为13.8%、13.1%、13.2%;2种酶解处理驴皮熬制的阿胶色红、气味芳香,与鲜驴皮熬制的阿胶相比色泽更鲜亮。2种酶解处理驴皮熬制阿胶的水分、灰分、水不溶物均符合并优于《中国药典》2015版一部中的质量标准,L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸含量也远高于《中国药典》2015版一部中的质量标准。[结论]低温条件下(4℃),1%碱性蛋白酶和1%中性蛋白酶可对醒发干驴皮进行有效酶解脱毛,且脱毛后驴皮熬制的阿胶质量符合《中国药典》2015版一部标准。

腐乳在发酵过程中脂肪酶活力变化研究

研究了雅致放射毛霉脂肪酶的酶学特性及其脂肪酶活力在腐乳发酵过程中的变化,和对脂肪水解作用的程度。研究发现,雅致放射毛霉脂肪酶的最适作用温度为40℃,最适pH为7.0。对腐乳在发酵不同阶段的pH,酸值,脂肪含量以及脂肪酶活力的研究中发现,腐乳在毛坯阶段其脂肪酶活力最高,自盐坯阶段起,脂肪酶活力一直呈下降的趋势。而脂肪含量在白坯、毛坯和盐坯(扣除盐分重量)阶段基本保持不变,在后期发酵阶段呈现下降的趋势。pH随着后期发酵的进行呈现出下降的趋势,脂肪酸值则呈现出相反的趋势,逐步上升。

微小毛霉凝乳酶基因定点突变及其酶学性质

微小毛霉凝乳酶具有很高的凝乳酶活力,是牛凝乳酶的潜在替代品,但由于微小毛霉凝乳酶蛋白水解活力过高,不利于干酪生产应用,所以本研究探索利用定点突变技术对微小毛霉凝乳酶基因进行分子改造,降低其蛋白水解活力及热稳性,提高凝乳活力。结果表明,凝乳酶肽链第186位由甘氨酸(Gly)突变为天冬氨酸(Asp)(即G186D)后,突变子凝乳活力显著提高,热稳定性有所降低;13位点由谷氨酸(Glu)分别突变为天冬氨酸(Asp)和谷氨酰胺(Gln)(即E13D,E13Q)后,突变子蛋白水解活力明显降低,同时凝乳活力稍有降低;第101位点由甘氨酸(Ala)突变为苏氨酸(Thr)(即A101T),突变子凝乳活力稍有提高,水解活力及热稳定性没有明显变化。此外,双重突变子,即101位点和186位点同时突变的突变子(即A101T/G186D)表现型与186单位点突变相一致,凝乳活力显著提高,热稳定性显著降低;186位点和13位点同时突变的突变子G186D/E13D和G186D/E13Q与13位点单点突变相比,凝乳活力有所提高,水解活力显著降低。

酶解毛血蛋白肽代替肠膜蛋白粉、鱼粉对保育猪生产性能的影响

为研究酶解毛血蛋白肽代替肠膜蛋白粉、鱼粉对保育猪生产性能的影响,笔者通过二次试验进行研究。试验一选择保育猪72头,根据性别、体重和遗传基础对等的原则随机分为2组,每组3个重复,每个重复12头。对照组日粮中含3.0%肠膜蛋白粉,试验组用1.5%酶解毛血蛋白肽代替3.0%肠膜蛋白粉,各组营养水平一致,试验期20 d。结果表明:与对照组相比,试验组保育猪的生产性能无显著差异(P>0.05)。试验二选择保育猪96头,根据性别、体重和遗传基础对等的原则随机分为3组,每组4个重复,每个重复8头。对照组饲喂含1.0%肠膜蛋白粉+1.6%鱼粉的对照日粮;试验1组用0.5%的酶解毛血蛋白肽代替1.0%的肠膜蛋白粉;试验2组用1.0%的酶解毛血蛋白肽代替1.0%的肠膜蛋白粉和1.0%的鱼粉,各组营养水平一致,试验期25 d。结果表明:保育猪日粮中用0.5%的酶解毛血蛋白肽代替1.0%的肠膜蛋白粉,或用1.0%的酶解毛血蛋白肽代替1.0%的肠膜蛋白粉和1.0%的鱼粉对保育猪的生产性能无显著影响(P>0.05),且能够降低饲料成本。

Intestinal endotoxemia plays a central role in development of hepatopulmonary syndrome in a cirrhotic rat model induced by multiple pathogenic factors

AIM: To characterize the correlation between severity of hepatopulmonary syndrome (HPS) and degree of hepatic dysfunction,and to explore how intestinal endotoxemia (IETM) affects the development of HPS in cirrhotic rats. METHODS: Male Wister rats were fed with a diet containing maize flour,lard,cholesterol,and alcohol and injected subcutaneously with CCl4 oil solution every two days for 8 wk to induce typical cirrhosis and development of HPS. The animals were also given a nitric oxide (NO) production inhibitor,Nω-nitro-L-arginine methyl ester (L-NAME) intraperitoneally,and an iNOS inhibitor,aminoguanidine hydrochloride (AG) via gavage daily from the end of the 4th wk to the end of the 6th or 8th wk,or a HO-1 inhibitor,zinc protoporphyrin (ZnPP) intraperitoneally 12 h prior to killing. Blood,liver and lung tissues were sampled. RESULTS: Histological deterioration of the lung paralleled to that of the liver in the cirrhotic rats. The number of pulmonary capillaries was progressively increased from 6.1 ± 1.1 (count/filed) at the 4th wk to 14.5 ± 2.4 (count/filed) at the 8th wk in the cirrhotic rats. Increased pulmonary capillaries were associated with increased blood levels of lipopolysaccharide (LPS)(0.31 ± 0.08 EU/mL vs control 0.09 ± 0.03 EU/mL),alanine transferase (ALT,219.1 ± 17.4 U/L vs control 5.9 ± 2.2 U/L) and portal vein pressure. Compared with normal control animals,the number of total cells in bronchoalveolar lavage fluid (BALF) of the cirrhotic rats at the 8th wk was not changed,but the number of macrophages and the ratio of macrophages to total cells were increased by nearly 2-fold,protein expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) started to increase significantly at the 4th wk,and reached its peak at the 8th wk in the lung of cirrhotic rats. The increase of iNOS expression appeared to be quicker than that of eNOS. NO2-/NO3-was also increased,which was correlated to the increase of iNOS (r = 0.7699,P < 0.0001) and eNOS (r = 0.5829,P < 0.002). mRNA expression of eNOS and iNOS was highly consistent with their protein expression. CONCLUSION: Progression and severity of HPS as indicated by both increased pulmonary capillaries and histological changes are closely associated with LPS levels and progression of hepatic dysfunction as indicated by increased levels of ALT and portal vein pressure. Intestinal endotoxemia plays a central role in the development of HPS in the cirrhotic rat model by inducing NO and/or CO.

Mechanism and Application of Enzyme Treatment on Wool

Protease treatments on wool have its inherent defect in shrink-proofing because protease will decompose wool through cell membrane complex （CMC）. In order to solve this problem, mechanism of enzyme treatments on wool was adequately analyzed and possible enzyme applications were discussed. The mechanism of enzyme treatment on wool was analyzed through weight loss, strength, scanning electron microscopy （SEM）, electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis （ SDS- PAGE）, and amino acids composition. Based on the results, a possible novel multifunetional enzyme treatment on wool to achieve shrink-resistance was proposed. In order to shorten enzyme treatment time, the rate of proteolysis of wool was investigated. Considering the specificity of proteases, wool composition, and structure of dyes, a better enzyme pretreatment before dyeing to get better dyeabflity and lower dyeing temperature was discussed.

Enzymatic Treatment for Shrink-proofing of Wool

An alkaline protease was applied on wool knitted fabric in an attempt to improve its shrink resistance. The influence of process parameters was evaluated and the tensile properties, surface characteristics and whiteness of wool were also studied. The results showed that the action of the protease on wool was significantly enhanced by hydrogen peroxide pretreatment. Based on the preliminary test and subsequent factor analysis, an appropriate combined process was obtained. The SEM investigation of the surface characteristics demonstrated that the enzymatic action was not uniform. In addition to the antifelting effects, the enzymatic treatment could lead to an improved degree of whiteness.

A sulfotransferase specific to N-21 of gonyautoxin 2/3 from crude enzyme extraction of toxic dinoflagellate Alexandrium tamarense CI01

Sulfotransferase （ST） is the first enzyme discovered in association with paralytic shellfish poisoning （PSP） toxin biosynthesis in toxic dinoflagellates. This study investigates the ST activity m crude enzyme extraction of a toxic dinoflagellate species, Alexandrium tamarense CI01. The results show that crude enzyme can transfer a sulfate group from 3＇-phosphoadenosine 5＇-phosphosulfate （PAPS） to N-21 in the carbamoyl group of gonyautoxin 2/3 （GTX2/3） to produce C 1/C2, but is inactive toward STX to produce GTX5. The crude enzyme is optimally active at pH 6.0 and 15℃. The activity is enhanced by Co^2＋, Mg^2＋, Mn^2＋ and Ca^2＋ individually, but is inhibited by Cu^2＋. Moreover, the activity shows no difference when various sulfur compounds are used as sulfate donors. These results demonstrate that the ST specific to GTX2/3 is present in the cells of A. tamarense CI01 and is involved in PSP toxin biosynthesis. In addition, the ST from different dinoflagellates is species-specific, which explains well the various biosynthesis pathways of the PSP toxins in toxic dinoflagellates.

Synthesis and Antiinflammatory Activity of 2 (4 Methoxy benzylidene) 5 aminomethyl Cyclopentanone Derivatives

In this paper, ten new derivatives of 2 (4 methoxybenzylidene) 5 aminomethyl cyclopentanone were designed and synthesized The structures of all these ten title compounds have been confirmed by IR, 1 H NMR and elemental analysis The compounds have been examined for the antiinflammatory activity on carrageenin induced rat paw edema test Through chemical synthesis, we have confirmed that the amino exchange reaction proceeds by an elimination addition mechanism

Integration of Ru/C and base for reductive catalytic fractionation of triploid poplar

Lignin,which is the most recalcitrant component of lignocellulosic biomass,is also the most abundant renewable aromatic resource.Herein,reductive treatment of triploid poplar sawdust by the integration of catalytic Ru/C and a base,which afforded high yields of phenolic monomers from the lignin component and a solid carbohydrate pulp,is reported.The introduction of Cs_(2)CO_(3) led to the generation of C2 side‐chained phenols through the cleavage of C_(β)–O and C_(β)–C_(γ) bonds inβ–O–4 units in addition to C3 side‐chained phenols;the relationship between C2 and C3 was dependent on the base dosage.The reaction conditions,including base species,temperature,time,and H_(2) pressure,were optimized in terms of phenolic product distribution,delignification degree,and carbohydrate retention.The carbohydrate pulps generated from reductive catalytic fractionation in the presence of Cs_(2)CO_(3) were more amenable to enzymatic hydrolysis,indicating that this treatment of biomass constituted the fractionation of biomass components together with the breakdown of biomass recalcitrance.

Characterization and Expression Profiling of Genes Encoding the Gibberellin 3-oxidase in Dasypyrum villosum Dwarf Mutant

Gibberellin 3-oxidase catalyzes the conversion of inactive gibberellin(GA) species into GAs with biological activity and it is subjected to strict developmental controls in the life cycle of a plant. In this study, 33 gene sequences, encoding the gibberellin 3-oxidase(GA3ox) from Dasypyrum villosum and its dwarf mutant, were obtained. Each contained a 1 107 bp coding sequence(CDS) that encoded a putative protein containing 369 amino acids. The GA3ox protein showed 77% to 97% homology and shared the major conserved structural domains of GA3ox proteins with rice, sorghum bicolor, oat, barley, and wheat. Sequence alignment showed that there were 20 single nucleotide polymorphisms(SNPs) and 22 Insertion/deletions(In Dels) among these sequences, which could be divided into 2 haplotypes, haplotypes Ⅰ and Ⅱ. Haplotype Ⅰ was found in the wild type and was1 495 bp in length, and haplotype Ⅱ was found in the dwarf mutant and was 1 485 bp in length. The Q-PCR results showed that GA3ox was expressed in the leaves, roots, internodes, and stem nodes, and that there was a significant difference in the transcript level of the GA3ox between the wild type and dwarf mutant. The transcript levels of GA3ox in the leaves at the seedling stage, stem elongation stage and the heading stage, in the root and stem nodes at the stem elongation stage and in the internodes at the heading stage of the wild type, were significantly higher than those in the dwarf mutant. However, GA3ox expression in the rest of the wild type tissues at the 3 stages was slightly higher than or not different from the dwarf mutant.The results suggested that the wild type and mutant allele sequences of GA3ox in D. villosum showed 2 amino acid changes in exons and variations in the lengths of introns or the SNPs in introns, which most probably impaired the function of the enzyme,affected the GA3ox expression level, and eventually gave rise to dwarfing.

Manganese peroxidase production from cassava residue by Phanerochaete chrysosporium in solid state fermentation and its decolorization of indigo carmine

Bioconversion of lignocellulosic wastes to higher value products through fungal fermentation has economic and ecological benefits. In this study, to develop an effective strategy for production of manganese peroxidase(Mn P)from cassava residue by Phanerochaete chrysosporium in solid state fermentation, the stimulators of Mn P production were screened and their concentrations were optimized by one-at-a-time experiment and Box–Behnken design. The maximum Mn P activity of 186.38 nkat·g-1dry mass of the sample was achieved after 6 days of fermentation with the supplement of 79.5 mmol·L-1·kg-1acetic acid, 3.21 ml·kg-1soybean oil, and 28.5 g·kg-1alkaline lignin, indicating that cassava residue is a promising substrate for Mn P production in solid state fermentation. Meanwhile, in vitro decolorization of indigo carmine by the crude Mn P was also carried out, attaining the ratio of 90.18% after 6 h of incubation. An oxidative mechanism of indigo carmine decolorization by Mn P was proposed based on the analysis of intermediate metabolites with ultra-high performance liquid chromatography and gas chromatography tandem mass spectrometry. Using the crude Mn P produced from cassava residue for indigo carmine decolorization gives an effective approach to treat dyeing effluents.

Wastewater Treatment for Removal of Recalcitrant Compounds：A Hybrid Process for Decolorization and Biodegradation of Dyes

While conventional wastewater treatments for urban effluents are fairly routine and have proved highly effective,industrial wastewater requires more complex and specific treatments.This paper provides a technological strategy for removal of recalcitrant contaminants based on a hybrid treatment system.The model effluent containing a binary mixture of synthetic dyes is treated by a combination of a preliminary physicochemical stage followed by a biological stage based on ligninolytic enzymes produced by Phanerochaete chrysosporium.This proposal includes biosorption onto peat as pretreatment,which decreases the volume and concentration to be treated in the biological reactor,thereby obtaining a completely decolorized effluent.The treated wastewater can therefore be reused in the dyeing baths with the consequent saving of water resources.

Kinetic Dependences of Two-Stage Dilute Acid and Enzyme Hydrolysis of Paulownia tomentosa

The sugars potential ofPaulownia tomentosa is estimated by dilute acid pretreatment and cellulase hydrolysis. The kinetics of dilute （1%） sulfuric acid hydrolysis is studied at temperatures of 100 ℃, 120 ℃ and 130 ℃, while the kinetics of the subsequent enzyme hydrolysis is examined at a temperature of 50 ℃ and reaction time varied from 60 to 300 min using cellulase complex NS 50013 and β-glucosidase N S 50010. The reducing sugars formation is modeled as a pseudo-homogeneous first order reaction in view of the kinetics of dilute sulfuric acid hydrolysis. The results obtained indicate that the reaction proceeds in an energetically homogeneous system （E = const） providing identical accessibility of the reagent to the hydrolyzing sites （A = const）. The enzyme hydrolysis kinetics follows heterogeneous catalytic mechanism. The process is described by an exponential kinetic equation, which is well recognised in case of processes on uniformly inhomogeneous surfaces. The current rate decreases significantly probably because of exhaustion of the available active sites on the surface and steric hindrances due to the presence of lignin. This investigation provides information of importance for the fermentation step of the bio-ethanol production process.

Antihyperuricemic Effect of Ethanol Extract of Snake Fruit （Salacca edulis Reinw.） var. Bongkok on Wistar Male Rat

The aim of the study was to investigate antihyperuricemic effect of snake fruit （Salacca edulis Reinw.） var. Bongkok Wistar male rates. Antihyperuricemic investigation on Wistar male rats showed that administration of ethanol extract at doses of 200 mg/kg bw decreased serum uric acid level significantly compared to control group at hour 6 and 7 （P 〈 0.05） after inducing with potassium oxonate intraperitoneally simultaneously with uric acid orally. Whereas, administration of ethanol extract at doses of 100 mg/kg bw did not decrease serum uric acid level significantly different compared to control group at hour 6 and 7 （P 〈 0.05）. Determination of uric acid level in urine, administration of ethanol extract at a dose of 200 mg/kg bw, or probenecid as a standard drug, at a dose of 45 mg/kg bw increased excretion of urine uric acid level significantly different compared to control group in day of 7 （P 〈 0.05） after inducing with potassium oxonate intraperitoneally simultaneously with uric acid orally. However, increase of uric acid excretion by ethanol extract was lower compared to that of probenecid at a dose of 45 mg/kg bw. Mechanism of action of the ethanol extract as an antihyperuricemia has been proposed by inhibition of xanthine oxidase and finally decreased the synthesis of uric acid and increased the excretion of urine uric acid level.

Impact of Combination Use of 0.004% Travoprost and 2% Pilocarpine on Matrix Metalloproteinases Synthesized by Rabbit Ciliary Muscle:a Pilot Study

Objective To explore the impact of combination use of prostaglandin analogue and cholinergic agonists on main matrix metalloproteinases(MMPs)synthesized by albino rabbit ciliary muscle.Methods Normal adult albino rabbits were divided into the control group,2%pilocarpine group,0.004%travoprost group and travoprost plus pilocarpine group.Two rabbits in the control group were executed after treated with normal saline for one day.Two rabbits were separately executed on the 7th,14th and 24th day of the treatment in each drug treated group.In each subgroup ciliary muscle band of 4 eyes was taken and made into homogenate.The MMPs activities of 10 subgroups were assayed by zymography.Bands’intensity which represents the activity of MMPs was measured by the UltraViolet Illumination system.Results A bright band of MMP-1/2 was showed on each lane at the position corresponding to the molecular weight of 62 kD in the ciliary smooth muscles electrophoresis.When ion Zn and Ca was displaced by MMPs inhibitor EDTA,this bright band disappeared.Compared with the control group,MMP1/2 activity increased by 4.0%,4.1%and 14.0%after 7,14 and 24 days of pilocarpine treatment.Corresponding data was23.2%,61.7%and 111.5%in the travoprost group and 49.3%,68.0%and 88.4%in the travoprost plus pilocarpine group.Conclusions Pilocarpine has little effect on activity of MMP1/2.Travoprost can increase activity of MMP1/2 gradually.Activity of MMP1/2 is rapidly increased by pilocarpine combined with travoprost,but shows small change with the prolonged treatment.