Thermostable alkaline protease production from Bacillus pumilus D-6 by using agro-residues as substrates

Proteases due to their wide range of applications in biotechnological processes have been the??focus of intense research for many decades. However, from industrial?application view point most of the available proteases lack desired properties;?therefore, search for better and efficient thermostable alkaline proteases are?always on.?Bacillus pumilus?D-6, isolated from dairy plant soil sample, in the?current study produced protease which showed activity and stability at high?alkaline?pH (8 - 12) and high?temperatures (70。C- 100。C). Enzyme activity remained unfazed even in presence?of inhibitors like Pb2+and Hg2+which are considered?universal inhibitors of enzyme activity. Besides, the organism successfully?utilized crude agriculture based substrates as carbon and nitrogen source and?produced substantial enzyme titre.

Analysis of Molecular Weight Distribution and Antioxidant Activities of Cirrhinus molitorella Skin Collagen Hydrolysates

[Objective] The aim of this work was to identify molecular weight （MW） distribution and antioxidant activity of fish skin col agen hydrolysates. [Method] The MW distribution of hydrolysates was determined using both size exclusion chromatography and matrix-assisted laser desorption ionization time-of-flight mass spec-trometry （MALDI-TOF-MS）. Fish skin were treated by the alkaline protease 2709. [Result] The optional conditions for hyerolysis were time 3 h, temperature 55 ℃, pH 10.0, substrate concentration 80 g/L and E/S 4%. The results of both methods indi-cated that the molecular weight of col agen hydrolysates was from 400 to 1 800 Da, and the peptides’ molecular weight was less than 1 400 Da mostly. The reducing power and antioxidant/radical scavenging activity [1, 1-diphenyl-2-picryl-hydrazyl （DPPH） free radical scavenging activity] were determined. [Conclusion] The results reveal that the fishskin hydrolysate is a potential source of antioxidants.

Study on Enzymatic Hydrolysis of Gadus morrhua Skin Collagen and Molecular Weight Distribution of Hydrolysates

Process parameters on enzymatic hydrolysis and molecular weight （MW） distribution of collagen hydrolysates from Gadus morrhua skin were investigated. The optimal process parameters were obtained by the single-factor and orthogonal experiments. The molecular weight distribution of hydrolysates was determined using both Sephadex G25 partition and high speed liquid chromatography electricity spray mass spectrum （HPLC-ESI-MS）. Collagen hydrolysates were first gained by an alkaline protease ＂alcalase＂ for 3 h at temperature （50~C）, pH （10.0）, substrate concentration （75 g L-~）, and E/S （3%）. The molecular weight distribution of collagen hydrolysates ranged from 300 to 1 500 Da, and most of peptides were under 1 200 Da. Sephadex G25 partition and HPLC-ESI-MS should be successfully employed to determine the molecular weight distribution of collagen hydrolysates.

Effect of Danshao Huaxian Capsule (丹芍化纤胶事囊) on the Expression of Collagen Ⅰ, Ⅲ, and Caspase-3 in Hepatic Fibrosis Rats

Objective: To study the effect of Danshao Huaxian capsule (丹芍化纤胶囊,DSHX), a traditional Chinese medical prescription, on the expression of collagen (Col) Ⅰ, Ⅲ and cysteinyl aspartate specific proteases-3 (caspase-3) in CCl4-induced hepatic fibrosis in rats. And also it is to explore the mechanism of DSHX in anti-fibrosis. Methods: Eighty male Wistar rats were randomly divided into the normal control group (A), the model group (B), the un-treated model group (C), the low-dose-DSHX treated group (D) and the high-dose-DSHX treated group (E). Except those in Group A, all the other rats were made into hepatic fibrotic models by comprehensive processes including subcutaneous injecting of CCl4, and feeding them with alcoholic high-fat and low-protein diet for 8 weeks. Then the two DSHX-treated groups were treated respectively with low dose (0.5 g/kg) and high dose (1.0 g/kg) DSHX capsule by gastrogavage everyday for 8 weeks. At the end of the experiment, the liver index, levels of hyaluronic acid (HA) and alanine amin-otransferase (ALT) in serum, degree of hepatic fibrosis, and urinary excretion of hydroxyproline (Hyp) were measured, and the expression of Col Ⅰ , Col Ⅲ and caspase-3 in liver tissues were detected respectively by immunohistochemistric technique. Results: Compared with those in Group B and C, the two DSHX treated groups showed that the liver index, levels of serum HA and ALT and severity of hepatic fibrosis were all significantly lower, the urinary excretion of Hyp was significantly higher; the Col Ⅰ and Col-Ⅲ expression was lower (Col Ⅰ :1. 23±1.14,1. 07±0. 96 vs 4.18±2. 26, 3. 22±1. 44, P<0. 01;Col Ⅲ : 1. 31±0. 69, , 1. 09± 0.58 vs 3.04±0.62,2.23±0.58, P<0.05). At the same time, the expression of caspase-3 in Group E was fewer than Group B and C in hepatocytes (3. 09±0. 65 vs 9. 60±2. 32, 8. 82 ±1. 45, P<0.01),but it was extensively expressed in fibrous septal cells(4.52±0.87 vs 1.69±0.23,2.98±0.36, P<0.01). Conclusion: DSHX capsule shows certain therapeutic effect on hepatic fibrosis in rats, and the mechanism might be related with reducing Col Ⅰ and Col Ⅲ deposition, inhibiting hepatocyte apoptosis and promoting fibrous septal cells (mainly the activated hepatic stellate cells) apoptosis.

Modified enzymatic collagen digestion-mediated isolation of osteocytes

This study established a method for isolating large numbers of high-purity osteocytes from high-density bone.Bone fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA nine times,and the digested cells and bone chips(BC)were cultured,digested,and passaged when cells were fully grown.The types of cells obtained were identified by morphology,viable cell counts,alkaline phosphatase staining,and biochemical activity analyses,and specific osteocyte and osteoblast markers were evaluated by quantitative real-time polymerase chain reaction.Our results showed that among the cells obtained from the third digestion(fractions 7–9)of wild mice tibias and femurs and the remaining BCs,85%–90%of the cells were osteocytes.Moreover,their morphology was approximately one-tenth to one-fifth the size of osteoblasts,star-shaped or polygonal,with a dendritic structure,negative for alkaline phosphatase staining,and showed a high expression of dmp1 and sclerostin.Ninety percent of the cells in fractions 1–3 were osteoblasts,and were fusiform or polygonal shape.The activity of osteoblast-specific alkaline phosphatase and mRNA expression were high in this fraction,while the expression of osteocyte-specific dmp1 and sclerostin was not detected.In the second portion(fractions 4–6),a large number were osteoblasts,mixed with a small number of osteocytes,and had high alkaline phosphatase activity and osteocyte mRNA levels,a specific level of the osteocyte marker dmp1,and no sclerostin was detected.Osteocytes in daβcatot mice were also successfully isolated by this method,and we found that Wnt signaling increased the proliferation of these osteocytes.The proposed method can be used to culture osteocytes and osteoblasts of high purity and can be used for isolation and culture of these two kinds of cells from high-density bone,which provides an avenue for the study of osteocyte function in vitro.

Significance of plasma von Willebrand factor level and von Willebrand factor-cleaving protease activity in patients with chronic renal diseases

Background von Willebrand factor （vWF） mediates the initial capture of platelets to vascular subendothelium and is essential for platelet aggregation under high fluid shear stress as in arterial stenosis. On release from endothelial cells, vWF is rapidly cleaved by ADAMTS13/vWF-cleaving protease （vWF-CP）. We investigated the clinical significance of changes in plasma vWF and vWF-CP activities in chronic renal disease.Methods Plasma vWF and vWF-CP activities were measured using enzyme-linked immunosorbent assay （ELISA） and residual collagen binding assay respectively in patients with lupus nephritis （n=31）, primary nephritic syndrome （n=25）, diabetic nephropathy （n=45）, chronic glomerulonephritis （n=38） and 40 normal controls. The relation of their levels with pathological and renal status was analyzed.Results In all diseased patients the levels of vWF were significantly higher and vWF-CP activity significantly lower than the controls （both P〈0.01）. vWF in the four subgroups did not correlate with the stage of disease but correlated negatively with vWF-CP activity, vWF-CP activity was not changed two weeks after renal transplantation. Renal biopsy demonstrated that the vWF level in stage IV was higher than in stages II and III while vWF-CP activity was lower in patients with lupus nephritis. After eight-week treatment, the vWF level significantly decreased and the vWF-CP activity significantly increased in systemic lupus erythema, disease activity index 〈9, but not with index 〉9. Even though the vWF-CP activity was significantly lower in membranous nephropathy than in minimal change disease, mesangial proliferative glomerulonephritis or IgA glomerulonephritis, the vWF level was not significantly different. Conclusions The alterations of plasma vWF and vWF-CP activities were associated with different renal pathologies. Injury to endothelial cells and autoantibodies against vWF-CP activity may result in higher vWF level and lower vWF-CP activity in chronic renal disease and thus a mechanism for worsening of chronic renal disease and thrombosis.

Characterization of acid-and pepsin-soluble collagens from spines and skulls of skipjack tuna(Katsuwonus pelamis)

Acid-soluble collagen(ASC) and pepsin-soluble collagen(PSC) from the spine(ASC-SP and PSC-SP) and skull(ASC-SK and PSC-SK) of the skipjack tuna, Katsuwonus pelamis, were successfully isolated and characterized. The yields of ASC-SP, PSC-SP, ASC-SK and PSC-SK were(2.47 ± 0.39)%,(5.62 ± 0.82)%,(3.57 ± 0.40)%, and(6.71 ± 0.81)%, respectively, on the basis of dry weight. The four collagens contained Gly(330.2-339.1 residues/1 000 residues) as the major amino acid, and their imino acid contents were between 168.8 and 178.2 residues/1 000 residues. Amino acid composition, SDS-PAGE, and FTIR investigations confirmed that ASC-SP and ASC-SK were mainly composed of type I collagen, and had higher contents of high-molecular weight cross-links than those of PSC-SK and PSC-SP. The FTIR investigation also certified all the collagens had triple helical structure. The denaturation temperatures of ASC-SK, PSC-SK, ASC-SP, and PSC-SP were 17.8, 16.6, 17.6, and 16.5 °C, respectively. All isolated collagens were soluble at acidic pH(1-5) and lost their solubilities when the NaCl concentration was above 2%(W/V). The isolated collagens from the spines and skulls of skipjack tuna could serve as an alternative source of collagens for further application in food, cosmetic, biomedical, and pharmaceutical industries.

Evaluation of von Willebrand factor-cleaving protease activity in patients with thrombotic thrombocytopenic purpura

Background Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy. In this study we investigated the von Willebrand factor-cleaving protease (vWF-cp) activity deficiency in patients with TTP.Methods The plasma or serum vWF-cp activity was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) by detecting the residual collagen binding activity (R-CBA) of von Willebrand factor (vWF) before and after digestion by vWF-cp. Multimers of vWF in plasma of patients with TTP were also analyzed by SDS-agarose electrophoresis. Moreover,the serum vWF-cp activities were compared between the patients with TTP and those with tumors.Results The coefficient of variation for intra-batch and inter-batch of the assay were 3.60% and 8.35%. The plasma and serum vWF-cp activity in healthy individuals were (78.79±9.17)% (n=30) and (79.47±10.78)% (n=53),respectively,while the plasma vWF-cp activity in 5 patients with TTP was markedly decreased [(21.83±19.98)%,P <0.001]. The unusually large vWF multimers were observed in two plasma samples of the patients with TTP. Although the vWF-cp activities in patients with benign and malignant tumors were also decreased ( P <0.03 and P <0.001,respectively),they were relatively high in comparison with that of TTP patients ( P <0.001).Conclusion Measurement of the vWF-cp activity using R-CBA is a simple and rapid method for diagnosing TTP. The vWF-cp activity in patients with TTP was markedly lower than those of patients with tumors.

碱性蛋白酶酶解蛋白制备活性肽的质控研究

以胶原蛋白和大豆分离蛋白为主要研究对象,应用体积排阻色谱(SEC)结合多角度激光光散射(MALLS)及示差折光(RI)联用技术对碱性蛋白酶酶解蛋白制备肽段的过程进行了详细分析。在酶解过程中,不同来源的胶原蛋白(牛、鸡)和大豆分离蛋白在分子量分布、体系的分散度及粒径变化规律上表现出了典型的差异。结果表明,牛胶原蛋白在酶解开始时就迅速被降解产生了大量小分子肽段,鸡胶原蛋白相对于牛胶原蛋白在相同的酶解条件下更难产生小分子肽段,大豆分离蛋白随着酶的加入则是表现出了先聚集再降解的特性。SEC-MALLS技术可以有效把控蛋白酶解的过程以及体系中肽段分子量分布,该技术可以为活性肽产品的开发及品质检测提供依据。

沙丁鱼鱼鳞胶原蛋白肽的制备及其抗氧化活性研究

目的优化沙丁鱼(Sardinapilchardus)鱼鳞胶原蛋白肽的制备工艺和评价不同分子量大小胶原蛋白肽的抗氧化活性。方法以沙丁鱼鱼鳞为原料,通过酶工程结合单因素与响应面实验优化胶原蛋白肽制备工艺,采用超滤膜分级分离鱼鳞胶原蛋白肽,并通过体外自由基化学体系分析对比不同分子量大小(10、5、1 kDa)的胶原蛋白肽的抗氧化活性。结果采用碱性蛋白酶进行酶解鱼鳞,胶原蛋白肽的得率最高;最优酶解工艺参数为:碱性蛋白酶加酶量7526 U/g、料液比1:20(g/mL)、pH 10.5、酶解温度67℃、酶解时间4 h,此条件下鱼鳞胶原蛋白肽得率最高,达43.44%±1.59%。通过超滤获得的分子量小于1k Da的鱼鳞胶原蛋白肽对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基和羟基自由基(hydroxyl radical,·OH)的清除能力最强,其半抑制浓度(half maximal inhibitory concentration,IC_(50))分别为0.72 mg/mL和1.76 mg/mL;分子量为1~5 kDa的鱼鳞胶原蛋白肽对超氧阴离子自由基(superoxide radical,O^(2-)·)的清除效果最好,其IC50为1.45 mg/mL。结论碱性蛋白酶能够有效制备鱼鳞胶原蛋白肽,且小于1 kDa的鱼鳞胶原蛋白肽抗氧化活性最好。

复合酶酶解法制备中华草龟皮胶原蛋白肽的工艺优化

该研究探讨了不同种类蛋白酶对中华草龟皮胶原蛋白酶解液的水解度和DPPH自由基清除率的影响,并进一步研究了复合蛋白酶对胶原蛋白的酶解效果。此外,通过正交试验优化了复合酶酶解条件。结果表明,由胰蛋白酶和碱性蛋白酶按1∶1组成的复合酶对龟皮胶原蛋白进行酶解,可显著提高酶解液的水解度和DPPH自由基的清除率(P<0.05);优化的复合酶酶解工艺为复合酶添加量5000 U/g、酶解pH值7.5、酶解温度50℃、酶解时间3 h,在该酶解工艺条件下,水解度达(51.19±2.45)%,DPPH自由基清除率达(51.78±2.21)%。影响复合酶酶解效果的主次顺序为酶添加量>酶解pH值>酶解时间>酶解温度。

酶解法制备澳洲坚果蛋白肽工艺优化研究

研究主要针对澳州坚果粕展开,依次选用了碱性蛋白酶、中性蛋白酶、复合蛋白酶、木瓜蛋白酶和风味蛋白酶进行研究分析,参考蛋白提取率和水解度的数据指标来确定最优的蛋白酶种类。以筛选出的最优蛋白酶进行了单因素实验,以确定最佳的添加量、原料浓度和酶解时间。借助响应面Box-Behnken中心组合实验来优化酶解工艺的过程。研究结果表明,碱性蛋白酶解澳洲坚果的蛋白提取率和水解度分别为48.30%和17.33%,显著高于其他4种蛋白酶。基于单因素实验和Box-Behnken响应面的中心组合实验的研究结果,在碱性蛋白酶中,各种因素对于澳洲坚果粕蛋白提取率和水解度存在着极显著的影响(P<0.01),而这些影响因素的优先级分别是:酶的添加量>底物的浓度>酶解的时长。得出了对澳洲坚果粕蛋白进行酶解的最佳工艺参数:底物质量浓度为8 g/100 mL,酶添加量为1600 U/g,酶解时间2 h。在此条件下,蛋白的提取率和水解度分别能达到56.527%和18.658%。

中性蛋白酶辅助提取天麻淀粉的工艺优化

以新鲜天麻为原料、中性蛋白酶为酶解剂,通过单因素和正交试验研究料液比、酶解时间、酶解pH、酶添加量和酶解温度对天麻淀粉中蛋白质残留率和天麻淀粉得率的影响,并确定最佳提取工艺。结果表明:天麻淀粉提取最佳工艺为料液比1∶7(g/mL)、酶解时间2.5 h、酶解温度35℃、酶添加量0.8 mg/g、酶解pH 7.0,在此条件下天麻淀粉得率为12.49%,蛋白质残留率为0.0068%。

黑木耳胶原蛋白酶法提取及分离纯化研究

为获得优质的黑木耳胶原蛋白,本研究通过单因素实验和响应面法对碱性蛋白酶法提取黑木耳胶原蛋白的工艺条件进行优化,并采用盐析、DEAE-52离子交换层析、Sephadex G-75凝胶过滤等方法对黑木耳胶原蛋白进行分离纯化。结果表明:在酶解温度55℃、pH 8.5条件下优化得到的黑木耳胶原蛋白最佳提取条件为:碱性蛋白酶(2240 IU/mg)添加量7%、料液比1∶68 g/mL、酶解时间2.0 h,此条件下,黑木耳胶原蛋白得率为0.65%;最佳盐析条件为:4 mol/L NaCl、24 h、15℃;最佳DEAE-52分离条件为:洗脱时间160 min、上样浓度300μg/mL、上样pH 7.2、NaCl洗脱浓度0.2 mol/L;经Sephadex G-75凝胶过滤层析后胶原蛋白纯度可达到72.6%,胶原蛋白回收率74.3%。因此,碱性蛋白酶法及盐析、DEAE-52、Sephadex G-75层析可有效提取纯化黑木耳胶原蛋白,为黑木耳胶原蛋白的深入研究及应用开发提供了理论依据。

酶解法制备土霉素菌渣吸附剂的研究

针对土霉素菌渣存在危险处置及不合理利用等问题,采用酶解法制备了土霉素菌渣吸附剂,以实现菌渣的资源化利用。采用高温高压蒸汽预处理菌渣,破坏菌丝体细胞壁和细胞膜结构;采用风味蛋白酶对菌丝体进行酶解处理,使菌丝体中的蛋白质、多肽等成分水解溶出,保留细胞多糖框架结构用作吸附剂的制备。结果表明,菌渣经酶解处理后菌丝分支变短、透光率提高;主要官能团没有发生改变;制备的菌渣吸附剂对亚甲基蓝的吸附率达到78.29%,比原菌渣的吸附量提高了1.44倍,达到了传统吸附剂活性炭的91.14%。

中药渣饲喂对蚯蚓生长性能及消化酶活性的影响

为解决畜禽粪污和药渣对环境污染的问题,探索中药渣和牛粪资源化利用的新途径,试验选取平均体质量为239.40 mg、平均体长为5.51 cm的健康蚯蚓,随机分成A、B、C和D组,每组设3个重复,在发酵牛粪的基础上分别添加0、10%、20%、30%的五味健脾颗粒药渣,室外饲养28 d。饲养0、28 d时,每组随机取蚯蚓9条,测量其体质量和体长;饲养0、7、14、21、28 d时每组随机取蚯蚓9条,提取蚯蚓粗酶液,用酶联免疫吸附法(ELISA)检测蛋白酶、纤维素酶和磷酸酶活性。饲养28 d时,与A组相比,C组蚯蚓体质量和体长显著增加,增重率提高55.50个百分点,体长增加1.83 cm;C组在饲养28 d时蛋白酶活性、纤维素酶活性和磷酸酶活性达到最高,且显著高于对照组(P<0.05)。以上结果表明,五味健脾颗粒药渣促进了蚯蚓生长发育,增强了消化酶活性,添加20%药渣效果最好。

碱性蛋白酶提取豆渣中不溶性膳食纤维的研究

采用碱性蛋白酶酶解提取豆渣中不溶性膳食纤维,在单因素试验的基础上,采用响应面法优化提取工艺,并对其持水力、持油力和溶胀性进行测定。结果表明:最佳工艺条件为酶解pH 9.0、酶解温度51℃、酶解时间4.1 h、酶添加量8250 U/g,在此条件下豆渣中不溶性膳食纤维含量为91.35%;不溶性膳食纤维的持水力、持油力及溶胀性分别为8.79 g/g、7.96 g/g、14.99 mL/g,与豆渣相比,分别提高了76.51%、92.74%、173.04%。

以罗非鱼加工废弃物为原料生产鱼鲜酱油的工艺研究

为提高罗非鱼加工废弃物(鱼残)的应用价值,以鱼残糜、虾壳粉和麸皮为原料,接种米曲霉(Aspergillus oryzae)CICC2066进行固体发酵制曲,再与鱼残糜进行高盐液体后发酵制备鱼鲜酱油。研究制曲时间、原料质量比和加水量对曲料中性蛋白酶活力的影响,采用三种不同预处理的鱼残糜,分别接种不同比例曲料,比较鱼鲜酱油中氨基酸态氮含量和感官评分。结果表明,制曲最佳条件为制曲时间44 h,鱼残糜、虾壳粉与麸皮质量比4∶5∶5,加水量50%。在此优化条件下,曲料中性蛋白酶活力达到1 330 U/g。采用鱼残糜加复合蛋白酶为原料,添加40%曲料进行后发酵,制得的鱼鲜酱油氨基酸态氮含量为1.37 g/100 mL,感官评分为95分。

实时荧光定量PCR法检测外源性DNA在重组Ⅲ型人源化胶原蛋白冻干纤维中的残留

目的对实时荧光定量PCR(qPCR)法检测重组Ⅲ型人源化胶原蛋白冻干纤维中的外源性DNA残留量进行探究和分析,确定该方法用于DNA质量控制的可行性。方法采用宿主细胞残留DNA样本前处理试剂盒(磁珠法)提取DNA,利用E.coli残留DNA检测试剂盒(PCR-荧光探针法)进行扩增反应,绘制标准曲线,建立重组Ⅲ型人源化胶原蛋白冻干纤维中外源性DNA残留量的qPCR检测方法。结果对8批重组Ⅲ型人源化胶原蛋白冻干纤维进行测定,外源性DNA残留量在0.03~300 pg/μl内,重复性良好,均远低于每剂10 ng,线性良好(R^(2)>0.99),回收率均在50%~150%之间。结论该方法可用于重组Ⅲ型人源化胶原蛋白冻干纤维中外源性DNA残留量的定量测定。

pH值渐变条件下双酶协同水解猪皮制备胶原蛋白寡肽的研究

本文研究了在没有外加碱的pH渐变条件下,用枯草杆菌碱性蛋白酶(Alcalase 2.4L)和黑曲霉酸性蛋白酶(3000U/g)双酶协同水解从猪皮胶原蛋白制备寡肽水解物的可行性。考察了单酶单因素水解条件、双酶加入方式对猪皮降解率和蛋白质水解度的影响,并在此基础上通过正交试验进一步优化出双酶一次性投料方案下水解猪皮的最佳条件:水解温度为60℃,碱性蛋白酶加入量为25μl/g蛋白,酸性蛋白酶加入量为4%(g/g),底物浓度为60g/L,水解时间为12h。在上述最佳条件下,新鲜猪皮的降解率可达94.7%,蛋白质的水解度可达17.7%,水解物中分子量小于1000(10个氨基酸以下的肽)的寡肽选择性达到了42.5%。结果表明,在不外加碱的条件下,采用双酶协同水解方法能够显著提高猪皮降解率,特别是蛋白质水解度。