一株产胶原蛋白酶细菌的鉴定及产酶条件优化

采用形态学特征、生理生化特征结合16S rDNA对一株产胞外胶原蛋白酶的菌株AL-13进行鉴定。获得安全、高效、高产量的生产胶原蛋白酶工艺。以胶原蛋白酶活性为指标,采用单因素实验优化温度、pH、接种量等产酶培养条件后,利用单因素结合响应曲面法优化产酶培养基。结果表明,AL-13鉴定为侧孢短芽孢杆菌(Brevibacillus laterosporus),最适产酶培养条件为:培养时间48 h、培养温度25 ℃、初始pH8.0、接种量6%(v/v),最适产酶培养基为:葡萄糖8.14 g/L、牛肉膏11.63 g/L、氯化钙0.17 g/L、磷酸氢二钾2.08 g/L、氯化钠9.48 g/L,在该产酶条件下胶原蛋白酶活力预测值为154.89 U/mL,验证结果显示酶活为(153.06±3.73) U/mL,此结果与预测值接近,相对误差为0.04%。本实验成功优化了一株产胶原蛋白酶细菌的产酶条件,产酶条件优化后的酶活(153.06 U/mL)较优化前(16.14 U/mL)提高了9.5倍。     

胶原蛋白酶酶解牛骨胶原蛋白的动力学研究

针对从细菌中提取的胶原蛋白酶酶解牛骨胶原蛋白的动力学性质进行研究。通过全波长扫描和SDS-PAGE电泳,对酶解的动态变化及其多肽分子量的变化进行了研究,考察了温度、酶用量、底物浓度等因素对酶解动态的影响,并对酶解的过程动力学进行了分析。结果说明:胶原蛋白酶酶解牛骨胶原蛋白得到的多肽分子量分布不均匀,分子量大小随反应时间的增大而减小;在一定范围内,增大酶浓度、提高温度可使酶解反应更彻底。     

Aeromonas sp.F3胶原酶性质的研究

海洋污泥中筛选得到的Aeromonas sp.F3所产的胞外酶对胶原蛋白有水解作用。以海洋微生物Aeromonas sp.F3为酶源,采用单因素试验法对其所产的胶原酶性质进行了研究,包括该胶原酶的酶解条件及热稳定性,并对该胶原酶水解鱼皮的效果进行了分析。结果表明,微生物Aeromonas sp.F3源胶原酶在50℃热处理40min之后明显失活,该胶原酶的最适反应温度为40℃,最适pH为8.6,金属离子Ca2+在0.5mmol/L时对酶有激活作用。该酶在其最适条件下,对鱼皮胶原有显著水解效能,其水解产物的分子质量在30ku以下。     

产骨胶原蛋白酶菌的筛选及发酵条件优化的研究

经初筛和复筛从骨骼厂土样中筛选出高产骨胶原蛋白酶的菌种MBL1,针对其产酶的最佳菌龄和发酵条件进行优化研究.结果表明,最佳接种菌的种龄选用培养24h,接种量为5%,起始pH值为7.0,150ml三角瓶最佳装液量20ml,发酵温度32℃.且在优化后的发酵条件下,该菌产酶酶活较优化前酶活提高了35%,达到49.83U/ml.     

胶原蛋白酶产生菌的筛选及初步鉴定

从养殖场附近淤泥中采样,分离得到20株胶原蛋白水解活性的菌株.经活性平板初筛、摇床复筛及酶活测定,筛选得到1株胶原蛋白酶活性为28.70U/mL的菌株.通过对该菌株16S rDNA的blast检索及序列分析表明,该菌株与蜡状芽孢杆菌属有99％以上的同源性,结合生理生化实验结果,经形态观察、生化试验,初步鉴定该菌株为蜡状芽孢杆菌.     

Real-time enzymatic degradation of human dentin collagen fibrils exposed to exogenous collagenase: an AFM study in situ

Objective: This study was carried out to observe the enzymatic degradation of human dentin collagen fibrils exposed to exogenous collagenase in situ using atomic force microscopy, to understand the characteristics of the enzymatic degradation of collagen fibrils on dentin specimens. Methods: Polished dentin specimens from caries‐free third molars were etched with citric acid, and then treated with an aqueous solution of 6.5% NaOCl for 120 s. The specimen was then put into a fluid cell and treated with a mixed solution of collagenase I (MMP‐1) and collagenase II (MMP‐8) for 9 h. AFM with contact mode was performed in situ to monitor the enzymatic degradation process of the dentin collagen fibrils. The distinctly topographic changes of the dentin surface were recorded continuously during different stages of the enzymatic degradation process. Results: The mixed solution of exogenous collagenase I and collagenase II could degrade dentin organic matrix (mainly collagen) efficiently, and the structures of dentin substrate were clearly exposed. Conclusion: It is possible to carry out real‐time observations on the enzymatic biodegradation process of human dentin collagen fibrils on dentin specimens with atomic force microscopy in situ. By this means, the fine structures of the etched dentin substrate were clearly revealed, possibly contributing to the related study of human dentin in vitro.     

The impact of collagen on softening of grass carp (Ctenopharyngodon idella) fillets stored under superchilled and ice storage

The objective of this work was to study the impact of collagen on softening of grass carp (Ctenopharyngodon idella) fillets during chilled storage. The fillets were stored under superchilling (?1.5 ± 0.2 °C) and with ice (0.2 ± 0.1 °C) for 21 days, and texture properties, collagen and the related enzyme activities were measured. Results showed that firmness and collagen content were strongly influenced by storage temperature and time. Fillet firmness decreased by 32.3% (superchilling) and 49.6% (ice stored) of the initial values after 3 days of storage, respectively. Total collagen content decreased with time, but different collagen fractions showed variations. Collagen degraded to different extents depending on storage conditions as indicated by SDS‐PAGE and amino acid analysis. In addition, collagenase activity declined significantly during the first 3 days, followed by a slow increase. This study demonstrated that collagen degradation was involved in grass carp fillet softening and provided useful information for fillet quality improvement.     

Evaluation of antioxidant and inhibitory activities for different subclasses flavonoids on enzymes for rheumatoid arthritis

Antioxidant activities of flavonoids were decreased in the order of flavonols > flavanones > flavones. Inhibitory intensities for hyaluronidase and collagenase reaction differed clearly according to flavonoid subclasses. Kaempferol, quercetin, myricetin, and rutin in flavonols inhibited hyaluronidase reaction specifically, while apigenin, luteolin, baicalin, and baicalein in flavones showed specific inhibition to collagenase reaction. In addition, the flavonoids, except baicalin and catechin, inhibited potently LPS-induced nitrite production in a dose-dependent manner, which might be mainly due to the suppression of inducible nitric oxide (NO) synthase. Quercetin and luteolin showed the strongest inhibitory activities on 15-lipoxygenase (LOX), and quercetin showed relatively potent inhibition on cyclooxygenase-1 (COX-1) reaction. Otherwise, all tested flavonoids possessed the inhibitory activity to COX-2 reaction, and especially luteolin, kaempferol, hesperetin, and naringin showed relatively the potent inhibition on COX-2 reaction. This report elucidated the anti-inflammatory activities, such as the antioxidant property, inhibition of NO production, and inhibition of inflammatory enzymes (hyaluronidase, collagenase, LOX, and COXs) of several subclass flavonoids.     

Structure and secretory activity of cultured chondrocytes from patients with osteoarthritis.

Cartilage samples were taken from OA patients in order to describe and quantify pro-inflammatory mediators. Samples were cultured under aseptic conditions in Dulbecco's modified Eagle medium at 37 degrees C for 10 days. Control samples, taken from non-inflammatory cartilage, were cultured under the same conditions. The levels of NO(-)2 and NO(-)3 were measured in the supernatant using a spectrophotometric assay. The activity of MMP-1 was quantified by ELISA. The concentration of NO(-)x was 47.3 +/- 4.1 microM in the OA cartilague and 10.7 +/- 1.8 microM in the controls. The average MMP-1 activity was 3,650 +/- 387 ng/ml in the OA cartilage and 2,150 +/- 190 ng/ml in the control samples. These increased values of MMP-1 and NO(-)x observed in the OA cartilage suggest a higher catabolic activity. A morphological analysis of OA chondral tissue using light microscopy shows that the surface of the tissue is characterized by the presence of aggregated chondrocytes or "clones" but in the deeper areas isolated cells are found. These results could be a significant contribution towards the identification of biological markers indicating the presence of OA activity.     

溶组织梭状芽孢杆菌胶原蛋白酶的研究进展

胶原蛋白是哺乳动物体内含量最丰富的蛋白质之一，因具有特殊的三股螺旋结构而不易被普通蛋白酶水解。胶原蛋白酶可在生理条件下特异性识别切割胶原蛋白，在医药、食品等工业领域应用广泛，其中以源自溶组织梭状芽孢杆菌的胶原蛋白酶研究较多。本文主要对来自溶组织梭状芽孢杆菌的胶原蛋白酶（collagenase，Col）G和ColH的结构特征、水解机理、生产现状和实际应用等方面进行概括总结，以期为胶原蛋白酶的进一步发展和利用提供理论参考依据。