链球菌噬菌体透明质酸裂解酶的异源表达及酶学性质表征

透明质酸酶是一类能降解透明质酸的酶的总称，在生物医药及药物递送等多方面具有重要的应用价值。为了挖掘高活性、高底物特异性及高稳定性的透明质酸酶，对来自链球菌噬菌体的透明质酸裂解酶(HylP)进行了异源表达及酶学性质表征。结果表明HylP由337个氨基酸残基构成，是多糖裂解酶家族16(PL16)新成员，重组HylP可在大肠埃希菌BL21(DE3)中可溶性表达，纯化产率可达到30 mg/L，最适酶活反应条件为40℃、pH 6.0，且在35℃及pH 6.0～7.0的中性条件下较为稳定。HylP专一性作用于透明质酸，比活力为24 u/mg；该酶的米氏常数(K_m)为0.13 mg/mL，催化常数(k_cat)为13.80 s^-1，具有较好的催化活性。链球菌噬菌体来源的HylP具有中温、高特异性、高酶活的特性，在绿色制备功能性低分子透明质酸上有潜在应用价值。     

注射用交联透明质酸钠凝胶的制备及其体外抗酶降解性的研究

目的筛选交联反应条件,制备强抗酶降解的注射用交联透明质酸钠(HA)凝胶(HA-凝胶),并建立其体外抗酶降解性的测定方法。方法用1,4-丁二醇二缩水甘油醚(BDDE)交联HA制备HA-凝胶,对其体外抗酶降解性的测定方法进行确证;采用正交试验考察6个因素对交联反应的影响,选择最佳反应条件。结果透明质酸酶(HAas)对HA-凝胶体外抗酶降解性测定方法无干扰,HAas在100~500 U/mL与产物显色后的吸光度呈线性关系(r=0.998 8)。正交试验极差分析和方差分析显示HA的起始反应浓度、BDDE与HA的比例对交联反应具有显著性影响,在氢氧化钠浓度1%、HA平均相对分子质量1.2×106、反应温度50℃、BDDE与HA的比例为1∶10(g/g)、反应时间4 h和HA浓度为15%的反应条件下,可得到抗酶降解性最好的HA-凝胶。结论选择适宜反应条件可以制备得到体外抗酶降解性强的HA-凝胶,为进一步开发注射美容和整形产品奠定了基础。     

透明质酸合成酶的研究进展

人类透明质酸合成酶(Hyaluronansynthase，HAS)是一类在透明质酸(Hyaluronic acid，HA)合成过程中发挥重要作用的酶，可分为HAS1、HAS2、HAS3 3种。这3种合成酶均可催化合成相当水平的透明质酸，且在特定的生理和病理过程中有所表达。但是，不同的透明质酸合成酶在稳定性、活性、催化产物HA的分子量等各方面也存在着明显差异。文章就人类3种透明质酸合成酶(HAS1、HAS2、HAS3)的分子特征、生物学活性、临床应用以及目前研究中存在的问题等研究情况进行综述，为治疗与HA相关的疾病提供理论依据。     

透明质酸氟脲嘧啶耦合物的制备与研究

目的合成肿瘤靶向药物透明质酸氟尿嘧啶耦联物,并探讨最佳合成条件。方法①以透明质酸（HA）和5-氟尿嘧啶（5-FU）为原料,将5-FU通过4位上的氨基与HA片段上的羧基耦合,得到HA-FU耦联物,测定其产物的紫外光谱。②以HA-FU耦联产物量作为指标,考察反应试剂加入的顺序、反应温度、溶液pH等多个水平,并从中选出最佳实验条件。结果制备HA-FU耦联物的最佳条件是将5-FU滴入HA中,温度为20-30℃,pH为3～4。合成了透明质酸与5-氟尿嘧啶耦合物,呈乳白色粉末。结论通过实验研究,找到合成透明质酸氟脲嘧啶的最佳合成条件。     

海藻酸钠法固定化谷氨酸脱羧酶的研究

目的研究海藻酸钠法固定化谷氨酸脱羧酶的最适条件。方法以海藻酸钠为载体,分别研究了海藻酸钠浓度、CaCl2浓度、固定化时间和固定化酶的最适pH、最适温度、最适底物浓度及反复利用的稳定性。结果最适固定化条件为3%海藻酸钠、0.1 mol/L CaCl2、固定化时间6 h。固定化酶的最适pH为5.0、最适温度为42℃、最适底物浓度1%,反应进行4次后一半的酶活损失,底物转化率在90%以上。结论海藻酸钠法固定化谷氨酸脱羧酶可以很好的转化谷氨酸钠生成γ-氨基丁酸,具有实用的潜力。     

高分子量透明质酸的发酵研究

采用兽疫链球菌(Streptococcus zooepidemicus)进行分批补料发酵生产透明质酸(HA).结果表明:采用葡萄糖初始浓度为4%,分批补加葡萄糖,使其浓度控制在2%,可使HA的产量达到3.4g/L,平均分子量2.45×106Da.若加入0.75g/L尿苷一磷酸,HA的产量可提高20.6%,达4.1g/L,平均分子量2.87×106Da.     

连续监测法测定血清亮氨酸氨基肽酶的活性及临床应用

目的应用亮氨酰对硝基苯胺(Leu-pNA)为底物建立测定血清亮氨酸氨基肽酶(LAP)活性的连续监测法,并探讨其在肝病诊断中的临床意义。方法基于LAP催化水解Leu-pNA生成有色对硝基苯胺的原理,对pH值、底物浓度、反应时间等条件进行研究,并用该法检测临床各种肝病血清LAP活性。结果酶促反应最适pH为7.20,最适底物浓度为4.8 mmol/L,批内、批间平均变异(CV%)分别为3.68%、5.91%。临床检验表明,肝硬化、活动性肝炎患者的血清LAP活性均高于正常组,呈现显著性差异(P<0.05)。结论本法灵敏度高,结果准确,且操作简便,在肝脏疾病诊断中具有临床意义。     

红非鲫(Oreochromis niloticus)鱼皮胶原蛋白酶解条件的研究

目的研究单酶和复合酶水解红非鲫鱼皮胶原蛋白制备具有抑制血管紧张素转换酶(ACE)生物活性的寡聚肽的工艺条件。方法根据6种单酶的水解产物对ACE抑制作用的测定结果,确定了两种酶即菠萝蛋白酶和alcalase 2.4L碱性蛋白酶组成复合水解酶。采用正交试验确定了这两种酶单独水解鱼皮胶原蛋白的最适酶解条件。结果与结论菠萝蛋白酶和alcalase 2.4L蛋白酶的最适酶解温度,pH,酶/底物,时间,底物浓度分别为45℃,pH4.5,4000U.g-1,4h,6%和55℃,pH7.5,6000U.g-1,2h,4%。在此基础上,进行复合酶水解实验,结果表明先采用菠萝蛋白酶水解,再用alcalase 2.4L蛋白酶水解,效果更佳。采用该双酶复合水解工艺,所得水解产物的水解度为30.43%,体外对ACE的抑制率达68.6%。     

透明质酸家族成员抗肿瘤治疗的研究进展

透明质酸(HA)是一种存在于细胞周基质和细胞外基质的非硫酸化糖胺聚糖,在肿瘤的发展和转移中起着重要作用。HA家族成员(透明质酸合成酶、透明质酸受体、透明质酸酶)已被证实能促进肿瘤的生长、转移和血管生成,基于HA家族成员的抗肿瘤治疗已引起广泛关注。目前,针对HA家族成员的肿瘤治疗方法主要包括小分子抑制剂、抗体、靶向递送抗肿瘤药物等。     

pH值、温度、底物浓度对L1型金属β-内酰胺酶活性的影响

目的 研究pH值、温度、底物浓度对L1型金属β-内酰胺酶活性的影响.方法 将表达后的L1酶纯化,以亚胺培南为底物,分光光度法测定不同pH值和温度的酶活性及不同底物浓度下的酶反应初速度,观察不同pH值,温度及底物浓度对酶活性的影响.结果 L1酶的最适pH为8.2,L1酶在40℃时活性最高.在亚胺培南浓度不超过30μmol/L时,反应初速度随底物浓度增加而呈线性增加,当底物浓度超过100μmol/L时,酶活性反而受抑,初速度明显下降.结论 该结果对进一步研究该酶的结构及酶学特性有重要意义,并可为开发有效的L1金属β-内酰胺酶抑制剂提供理论指导.     

Hyaluronidases in Loxosceles intermedia (Brown spider) venom are endo-beta-N-acetyl-d-hexosaminidases hydrolases.

In studying Loxosceles venom, we detected degradation of purified hyaluronic acid (HA) and hydrolysis of purified chondroitin sulphate (CS) while neither dermatan sulphate, heparin or heparan sulphate were affected. In addition, with HA-degrading kinetic assays, we show that a hydrolase enzyme was involved in the HA cleavage. By use of the Reissig colorimetric reaction, we found that venom hyaluronidase is an endo-beta-N-acetyl-d-hexosaminidase that generates terminal N-acetylglucosamine residues upon cleavage of HA. Zymogram analysis of L. intermedia venom showed HA lytic activities at 41 and 43kDa, and, when CS was used as a substrate, zymograph experiments resulted in 41 and 43kDa lytic zones. Thus, these results support the hypothesis that the same molecules are involved in cleaving HA and CS residues. Experiments to compare L. intermedia electrostimulated venom and venom gland extract also demonstrated very similar HA lytic activity, suggesting again that hyaluronidases are self-components of Loxosceles spider venom instead of oral egesta contamination. HA degradation as a function of pH in these hydrolase enzymes showed no apparent activities at low or high pH, with optimal activity at 6.0-8.0 pH. Finally, we confirmed the cleaving action of the venom hyaluronidases on HA in the extracellular matrix of the dermis of rabbit by fluorescence reaction to HA and confocal microscope analysis. Thus, hyaluronidases type hydrolases endo-beta-N-acetyl-d-hexosaminidase are implicated as self-components of Loxosceles spider venom and can be involved in venom effects as spreading factors.     

Evaluation of hyaluronic acid-protein conjugates for polymer masked-unmasked protein therapy

Bioresponsive polymers may effectively be utilized to enhance the circulation time and stability of biologically active proteins and peptides, while reducing their immunogenicity and toxicity. Recently, dextrin-epidermal growth factor (EGF) conjugates, which make use of the Polymer-masked UnMasked Protein Therapy (PUMPT) concept, have been developed and shown potential as modulators of impaired wound healing. This study investigated the potential of PUMPT using hyaluronic acid (HA) conjugates to mask activity and enhance protein stability, while allowing restoration of biological activity following triggered degradation. HA fragments (Mw ～90,000g/mol), obtained by acid hydrolysis of Rooster comb HA, were conjugated to trypsin as a model enzyme or to EGF as a model growth factor. Conjugates contained 2.45 and 0.98% (w/w) trypsin or EGF, respectively, and contained <5% free protein. HA conjugation did not significantly alter trypsin's activity. However, incubation of the conjugate with physiological concentrations of HAase increased its activity to ～145% (p<0.001) that of the free enzyme. In contrast, when HA-EGF conjugates were tested in vitro, no effect on cell proliferation was seen, even in the presence of HAase. HA conjugates did not display typical masking/unmasking behavior, HA-trypsin conjugates exhibited ～52% greater stability in the presence of elastase, compared to free trypsin, demonstrating the potential of HA conjugates for further development as modulators of tissue repair.     

Evaluation of the physical and biological properties of hyaluronan and hyaluronan fragments

Hyaluronan (HA) has been extensively used for various medical applications, including osteoarthritis, tissue augmentation and ocular surgery. More recently, it has been investigated for use in polymer therapeutics as a carrier for drugs and biologically active proteins, thanks to its biodegradability, biocompatibility and inherent biological properties. Such biological functions are strongly dependent on HA's chain length, yet the molecular weight of HAs used in polymer conjugates varies widely and is inconsistent with its intended application. Therefore, this study aimed to determine the ideal chain length of HA to be used in polymer conjugates for enhanced tissue repair.HA fragments (M_w 45,000-900,000 g/mol) were prepared by acid hydrolysis of rooster comb HA and their physicochemical and biological properties were characterized. Such HA fragments had a highly extended, almost rod-like solution conformation and demonstrated chain length- and concentration-dependent viscosity, while exposure to HAase caused a rapid reduction in HA viscosity, which was most significant for the native HA. Initial HA hydrolysis rate by HAase varied strongly with HA chain length and was dependent on the formation of a stable enzyme-substrate complex. When normal human dermal fibroblasts were exposed to the different HA fragments for 72 h, only native (900,000 g/mol) HA reduced proliferation at 1000 μg/mL. Conversely, only the smallest HA fragment (70,000 g/mol) reduced the proliferation of chronic wound fibroblasts, at 1000 μg/mL. The 70,000 g/mol HA fragment also promoted the greatest cell attachment.These observations demonstrate that low molecular weight (70,000-120,000 g/mol) HA fragments would be best suited for the delivery of proteins and peptides with applications in chronic wound healing and paves the way for the rationalized development of novel HA conjugates.     

Hydrolytic kinetics of lithospermic acid B extracted from roots of Salvia miltiorrhiza

The hydrolytic kinetics of lithospermic acid B (LAB) extracted from the roots of Salvia miltiorrhiza (Chinese herb: danshen) was investigated by using reversed-phase high-performance liquid chromatography (HPLC) with UV-vis detection. The influences of initial drug concentration, pH and temperature on hydrolysis of LAB were studied in aqueous solutions. The results showed that initial concentration of LAB has no effect on the degradation rate at pH 2.0. The hydrolysis followed pseudo-first-order kinetics at 90 degrees C. The log k(obs)-pH profile indicated that the optimal stability range was at pH 2.0-5.0. The rate constant of overall hydrolysis as a function of temperature under the given conditions obeyed the Arrhenius equation. Analysis of the acid-induced degraded solution of LAB by liquid chromatography-mass spectrometry (LC-MS) revealed at least four degradation products [M-H](-) ion at m/z 197, 137, 537 and 537, respectively. Three of these degradation products, i.e. danshensu (DSU), protocatechuic aldehyde (PRO), and lithospermic acid, were further identified by comparing the retention times with standard samples. According to the structure of LAB and its hydrolysis behavior in solution, the other product was proposed to be the isomer of lithospermic acid.     

酶法制取黄粉虫蛋白复合氨基酸液的研究

目的探讨胃蛋白酶水解黄粉虫蛋白制取复合氨基酸液的工艺。方法以黄粉虫为原料,以碱法提取黄粉虫蛋白,采用胃蛋白酶水解黄粉虫蛋白制取复合氨基酸液。通过单因素试验探讨了底物浓度、pH值、酶浓度、水解温度及水解时间与水解率的关系,采用正交试验L16（4^5）优选水解工艺条件。结果最佳酶解工艺条件为：底物浓度为1∶5,pH值4.0,酶浓度1.5%,水解温度60℃,水解时间5 h。水解率达到24.15%。结论按最佳条件水解完毕后,通过酶灭活,中和,脱色等处理可得到黄色澄清的黄粉虫蛋白复合氨基酸液。所制得的复合氨基酸种类齐全,必需氨基酸含量高。     

光谱法测定多酚类化合物与玻璃酸酶的相互作用

目的:研究多酚类化合物与玻璃酸酶(Hyaluronidase,HAase)的相互作用。方法:利用荧光猝灭法和圆二色谱(CD谱)法比较几种不同的多酚化合物和HAase在25℃和pH7.4条件下相互作用的结合常数(Ka)值,以及比较不同温度下黄芩素与HAase相互作用的Ka值。结果:多酚类化合物能猝灭HAase的内源荧光,且黄芩素、汗黄芩素、黄芩苷比葛根素、白藜芦醇对HAase的内源荧光具有更强的猝灭能力,黄芩素和白藜芦醇分别与HAase作用显示不同CD谱。结论:多酚类化合物与HAase的相互作用为静态猝灭过程,黄芩素和白藜芦醇这2种多酚化合物与HAase作用具有不同的结合模式。     

Lepidepyrone, a new gamma-pyrone derivative, from Neolentinus lepideus, inhibits hyaluronidase

In the course of screening for hyaluronidase (HAase) inhibitory agents, a new gamma-pyrone derivative, lepidepyrone, C(8)H(10)O(5), was isolated from the cultured mycelium of the mushroom Neolentinus lepideus TMC-1102 as a major HAase inhibitory compound (IC(50) 3.3 mM). The structure of lepidepyrone was established on the basis of spectroscopic investigation.     

Degradation kinetics of L-glutamine in aqueous solution.

The degradation kinetics of L-glutamine (Gln) in aqueous solution was studied as a function of buffer concentration, pH and temperature. Stability tests were performed using a stability-indicating high-performance liquid chromatographic assay. The degradation product of Gln was 5-pyrrolidone-2-carboxylic acid. The reaction order for Gln in aqueous solution followed pseudo-first-order kinetics under all experimental conditions. The maximum stability of Gln was observed in the pH range from 5.0 to 7. 5. The pH-rate profile described by specific acid-base catalysis and hydrolysis by water molecules agreed with the experimental results. Arrhenius plots showed the temperature dependence of Gln degradation, and the apparent activation energy at pH 6.41 was determined to be 9.87 x 10(4) J mol(-1).     

反胶团酶促水解青霉素G制备6-APA

研究将青霉素酰化酶包埋于ＡＯＴ／异辛烷反胶团中水解青霉素Ｇ制备６－ＡＰＡ的工艺,探讨了各种操作条件对水解过程的影响。结果表明,在反胶团酶反应体系中,底物浓度提高了一倍左右,且在反应过程中不需滴碱调节ｐＨ值。在水相与反胶团体系各自的最适条件下分别进行水解反应实验,结果表明,青霉素酰化酶在反胶团体系中的酶活力高于水相酶活力,水解６ｈ 后其转化率可达７０％ 以上;与相应的水相酶促反应相比,转化率提高了３０％ 。