Isolation and Characteristics of Bacillus subtilis CN2 and its Collagenase Production

ABSTRACT: :
An isolated bacterium strain named CN2 found in Vietnamese fish sauce has been identified as Bacillus subtilis. In an enzyme-producing medium with 0% and 8% NaCl concentration, the CN2 strain produced the maximum collagenase activity, 3.07 U/ml and 2.60 U/ml. The strain also produced gelatinase, but the maximum activity was only 1.03 U/ml at 8 h of incubation time and prolonged more than 22 h. Bacillus subtilis CN2, grown slowly in a medium containing 12% NaCl, showed a decreased rate of collagenase activity with a maximum activity of 1.60 U/ml at 18 h of incubation time. The culture supernatant of CN2 strain digested a purified native collagen from rat tail tendon as well as αs-casein at Met¹²³-Lys¹²⁴ position. Therefore The culture supernatant of CN2 can be used to produce healthy foods.     

Clostridium Collagenase Impact on Zone of Stasis Stabilization and Transition to Healthy Tissue in Burns

Clostridium collagenase has provided superior clinical results in achieving digestion of immediate and accumulating devitalized collagen tissue. Recent studies suggest that debridement via Clostridium collagenase modulates a cellular response to foster an anti-inflammatory microenvironment milieu, allowing for a more coordinated healing response. In an effort to better understand its role in burn wounds, we evaluated Clostridium collagenase’s ability to effectively minimize burn progression using the classic burn comb model in pigs. Following burn injury, wounds were treated with Clostridium collagenase or control vehicle daily and biopsied at various time points. Biopsies were evaluated for factors associated with progressing necrosis as well as inflammatory response associated with treatment. Data presented herein showed that Clostridium collagenase treatment prevented destruction of dermal collagen. Additionally, treatment with collagenase reduced necrosis (HMGB1) and apoptosis (CC3a) early in burn injuries, allowing for increased infiltration of cells and protecting tissue from conversion. Furthermore, early epidermal separation and epidermal loss with a clearly defined basement membrane was observed in the treated wounds. We also show that collagenase treatment provided an early and improved inflammatory response followed by faster resolution in neutrophils. In assessing the inflammatory response, collagenase-treated wounds exhibited significantly greater neutrophil influx at day 1, with macrophage recruitment throughout days 2 and 4. In further evaluation, macrophage polarization to MHC II and vascular network maintenance were significantly increased in collagenase-treated wounds, indicative of a pro-resolving macrophage environment. Taken together, these data validate the impact of clostridial collagenases in the pathophysiology of burn wounds and that they complement patient outcomes in the clinical scenario.     

Degradation in muscle fibre-connective tissue junctions in the spotted trevalla (seriolella punctata) examined by scanning electron microscopy

The junctions between the muscle fibres and the connective tissues of the myocommata in the spotted trevalla (Seriolella punctata Forster) were investigated by scanning electron microscopy. In prerigor muscle, the fine collagen fibrils which arise from the myocommata to form the muscle cell envelope were evident. After the fish were stored several days in ice, progressive deterioration was observed in these fibrils. The structure and the degradation was similar in nature to that reported previously in blue grenadier.     

Collagenase Effect on Thermal Denaturation of Intramuscular Collagen

We have investigated the action of a collagenase from Clostridium histolyticum on the thermal denaturation parameters (AH and initial temperature of denaturation [qt]) of bovine intramuscular collagen. Collagens exhibiting various degrees of reticulation were extracted from calves, steers and cull cows pectoralis profundis muscle. Collagenase treatment induced a decrease in the enthalpy of denaturation of calf and steer collagens inversely related to animal age, but no change was observed for cow's collagen. Irrespective of the degree of reticulation, collagen breakdown by the collagenase led to the appearance of another peak of denaturation starting at a lower temperature (55–57°C) than that of intact collagen (62–63°C).     

Encapsulation of all-trans retinoic acid into wheat ceramides micro vesicles enhanced influence on collagenase and tissue inhibitor of metalloproteinase-1 production by human skin fibroblasts

The influence of all-trans retinoic acid (RA), either in its free or encapsulated form into wheat ceramides (CER), on the production of collagenase (MMP-1), and tissue inhibitor of metalloproteinase (TIMP-1) by human skin fibroblasts (HSF) at early and late stages of their proliferative life span (PLS) was examined. The level of MMP-1 was elevated and that of TIMP-1 decreased in late as compared to early passage cells. All-trans retinoic acid significantly decreased and increased the secretions of MMP-1 and TIMP-1 respectively, in a dose-dependent manner, from 10-7 m to 10-5 m. Entrapment of RA into CER vesicles potentiated its effect on MMP-1 and TIMP-1 secretions by HSF, independently of cell passages. The extent of variations obtained on MMP-1 and TIMP-1 levels, when HSF culture medium was supplemented with 10-5 m RA, could be obtained using a 100-fold lower concentration of RA encapsulated into CER vesicles. CER had no effect on TIMP-1 and MMP production by HSF in culture, and simultaneous addition of CER and RA did not potentiate the effects of RA alone, indicating that formation of RA-CER liposomes was responsible for the enhancing effects. The rate of internalization of RA into HSF was increased when used in its CER encapsulated form. Therefore, the use of retinoid within CER potentiates its beneficial influence on the MMP-1:TIMP-1 imbalance with fibroblastageing.     

蜡样芽孢杆菌胶原蛋白酶基因的异源表达与活性分析

研究采用降解骨胶原蛋白的蜡样芽孢杆菌MBL13-U作为原菌种,并对蜡样芽孢杆菌MBL13-U进行全基因组测序,克隆获得胶原蛋白酶(ColM13)基因。将目的基因与大肠杆菌的表达载体pET30a进行连接,转入大肠杆菌宿主菌株BL21,获得降解骨胶原蛋白的工程菌pET30a-ColM13/BL21。通过SDS-PAGE测定异源表达的重组蛋白酶ColM13的分子质量,并测定不同诱导时间和诱导浓度对ColM13酶活性的影响,最终确定重组蛋白酶ColM13的酶活最适条件:6‰异丙基硫代半乳糖苷(IPTG,100 mmol/L),37℃诱导6 h,优化后酶活力最高为64.99 U/mL,较优化前提高4.24倍。通过水解圈试验、紫外光谱和扫描电子显微镜等方式验证,表明工程菌构建成功,这为我国畜禽骨骼资源的深度开发和综合利用提供了新的研究思路。     

牛骨胶原蛋白酶解工艺优化及结构特性分析

为研究前期纯化的特异性降解牛骨胶原蛋白的胶原蛋白酶(BSC)对牛骨胶原蛋白的酶解工艺,以水解度为响应值,在单因素试验基础上通过五元二次正交旋转组合试验,确定最佳酶解工艺为:反应温度46℃、牛骨胶原蛋白添加量5.14g/100mL、BSC添加量0.42g/100 mL、反应时间6h、pH 6.5,该工艺条件下水解度为34.98%。采用紫外光谱(UV)、荧光光谱(FS)、傅里叶红外光谱(FT-IR)、扫描电镜(SEM)对牛骨胶原蛋白及其产物进行结构特性分析。通过UV分析表明BSC酶的降解作用破坏了牛骨胶原蛋白的三股螺旋结构,游离出大量的氨基酸;FS分析表明牛骨胶原蛋白分子表面C═O、CONH_2、COOH逐步增多,胶原蛋白肽中生色基团分布也随之发生变化;FT-IR分析可知牛骨胶原蛋白经降解后的胶原蛋白肽的肽链上—NH_2~+—排斥作用逐渐减弱,主要以β-转角为主;SEM表明酶破坏了牛骨胶原蛋白的表面分子结构,使其变得疏松。     

Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.