融合自组装双亲短肽提高碱性果胶酶热稳定性

考察了N-端融合自组装双亲短肽（SAP）对PGL热稳定性的影响.将6种具有不同疏水性和柔性的SAP融合至Bacillus sp.WSHB04-02 PGL N-端,得到融合酶PGL-S1、PGL-S2、PGL-S3、PGL-S4、PGL-S5和PGL-S6.与PGL相比,融合酶的55℃半衰期（t1/2）提高9.69 ～ 72.03倍;PGL-S1比酶活提高1.5倍达到458.52 U/mL,其他融合酶比酶活下降38.27％ ～93.94％.此外,6种融合酶Km均较PGL显著下降;S1-PGL的kcat/Km提高3.52倍,其他融合酶kcat/Km则下降38％以上.所有SAP-PGL融合酶最适反应pH均在pH 9.4～10.6,符合其工业应用环境要求.上述结果表明,N-端融合SAP能有效提高PGL热稳定性和催化活性,获得的融合酶S1-PGL具有理想的应用前景.

聚唾液酸修饰CuZn-SOD及其形态和稳定性研究

聚唾液酸(Polysialic acid,PSA)因其更为有效的安全性,被公认为传统药用缓释材料——聚乙二醇(PEG)的理想替代材料。作者对蛋白质药物——铜锌超氧化物歧化酶(CuZn-SOD)的聚唾液酸修饰进行了初步研究。研究了修饰反应的主要条件:聚唾液酸经高碘酸钾氧化后生成的活化体,与超氧化物歧化酶以40∶1的摩尔比反应24h,可以达到理想修饰效果,生成的聚唾液酸修饰的超氧化物歧化酶衍生物(PSA-SOD)平均交联比达到了3.9±0.3。该衍生物经过SDS-PAGE电泳和原子力显微镜(AFM)检测测得其平均相对分子质量为90 000～100 000,分子粒径为10～15nm,约为天然超氧化物歧化酶的4倍。除此,PSA-SOD呈单颗粒包覆和多颗粒包覆两种显微形态。PSA-SOD进行稳定性试验表明,经聚唾液酸修饰的超氧化物歧化酶的酸碱稳定性、热稳定性和抗酶解稳定性都有了较大程度的提高。

Fe^(2+)和Fe^(3+)对菠萝蛋白酶活性和稳定性的影响

本文研究不同浓度的Fe^(2+)和Fe^(3+)对菠萝蛋白酶活性和60℃下酶稳定性的影响,以及通过圆二色谱检测Fe^(2+)和Fe^(3+)对酶构象的变化,并初步探究EDTA-2Na结合超滤膜法在菠萝蛋白酶制备工艺中清除铁离子的应用效果。结果表明:Fe^(2+)浓度在0~0.75 mmol/L范围内,对菠萝蛋白酶活性有促进作用,以0.5 mmol/L促进效果最佳,但浓度大于0.75 mmol/L时,抑制酶活,且随浓度增加,抑制程度增强。Fe^(3+)对菠萝蛋白酶只有抑制作用,抑制程度与Fe^(3+)浓度成正比,且Fe^(3+)对酶的抑制作用强于Fe^(2+)。60℃下,Fe^(2+)在浓度为0.5 mmol/L对菠萝蛋白酶的稳定性表现出促进作用,延长了酶的半衰期。Fe^(3+)对酶的稳定性呈现抑制作用。Fe^(2+)和Fe^(3+)浓度大于0.5mmol/L时,随离子浓度的升高,对酶稳定性的抑制作用增强。采用圆二色谱检测酶的二级结构表明:Fe^(2+)和Fe^(3+)对酶抑制作用表现为α-螺旋含量下降,β-折叠和β-转角含量稍下降,无规卷曲含量明显提高。EDTA-2Na结合超滤膜法除铁的效果很明显,铁含量(干重)从291.63 mg/kg降低到142.99 mg/kg,铁离子去除率达50.97%,其酶活也由597.27 U/mg上升到808.52 U/mg,酶活提高了26.13%。

一种来源于Bispora betulina的酸性木聚糖酶基因克隆及其酶学性质研究

利用简并PCR和TAIL-PCR技术从嗜酸性真菌Bispora betulina中克隆得到一个酸性木聚糖酶基因xyn11BB。该基因全长1 207 bp,含有三段内含子、一段外显子和一个终止密码子,编码由297个氨基酸组成的蛋白质,推测蛋白质含有一个CBM1结构域。将不带原基因信号肽编码序列的cDNA基因以正确阅读框架克隆到表达载体pPIC9上,并在毕赤酵母(Pichia pastoris)GS115中诱导表达,其表达活性达121.15 U/mL。重组蛋白经硫酸铵分级沉淀和阴离子交换柱纯化后达到电泳纯。对其酶学性质分析表明,重组木聚糖酶最适温度为50℃,最适pH为4.5,在酸性条件下具有良好的活性和稳定性,可作为饲料用酶的备选。

分子筛SBA-15固定化溶菌酶的研究

用分子筛SBA-15作为固定载体吸附溶菌酶(Lysozyme,Lyz)制成功能化材料SBA-15-Lyz。研究介质的溶菌酶浓度、温度、p H、Na^(+)浓度和吸附时间对SBA-15固定溶菌酶的影响。利用扫描电镜、透射电镜、傅里叶变换红外光谱和热重仪对SBA-15-Lyz进行表征,并测定其热稳定性与pH稳定性。结果显示:SBA-15对溶菌酶的固定容量与溶菌酶介质的浓度、吸附时间以及温度呈正相关,与盐离子浓度呈负相关。温度为35℃时达到最大固定容量691.04 mg/g。SBA-15吸附溶菌酶的行为符合Langmuir模型(R^(2)=0.978 6)与准二级动力模型(R^(2)=0.999 9)。将固定酶在100℃和pH 10.0的条件下分别孵育1 h后发现,固定酶具有比游离酶更强的热稳定性与pH稳定性,相对酶活分别保留43.40%和84.76%,有利于拓宽溶菌酶在食品杀菌领域的应用范围。

Activity and Stability of Arginine Deiminase for Producing L-citrulline

A novel Enterococcus faecalis strain designated N J402 was found with high activity of arginine deiminase （ADI）. The optimum condition for catalytic activity was determined in terms of temperature （about 40℃）, thermostability （available 37℃） and pH （6-7）. The effects of substrate and product concentration were studied. The effects of various metal ions added in reaction mixtures on the biocatalyst were investigated and ADI of N J402 was found to exhibit Co^2＋ dependence, different from previous reports. Surfactant, cetyl trimethyl ammonium bromide, was one of the most important keys for producing L-citrulline. The enzyme in resting cells possessed the quality of high stability for reuse.

Performance of a Pectinase from Bacillus Subtilis WSHB04-02 Used in Bioscouring of Cotton Fabrics

A pectinase produced by Bacillus subtilis WSHB04-02 isolated from soil with lyase activity operating at alkaline pH was studied. The Michaelis-Menten kinetic parameters of this newly isolated pectinase on different substrates, such as citrus pectin and polygalacturonic acid (PGA), were determined, and pectin proved to be the most suitable substrate. The effects of temperature and pH on pectinase activity and stability were also investigated. The optimal temperature for pectinase was 55℃ with a stable range of 45℃-55℃. In general, pectinase was pH insensitive and the stable pH ranged from 8.6 to 10.0. Ultimately the bioscouring effects of cotton fabrics using this pectinase were evaluated and some promising results were obtained.

State of Pepsin and Acidic Protease in Solid Phase System and the Factors Increasing Their Destabilization

The adsorption state and catalytic properties of pepsin and acidic protease from microorganisms Asp. awamori and Asp. oryzae were studied in solid phase system （in presence of sorsilen, DEAE- and CM-cellulose）. According to the results, adsorption capacity and catalytic activity of enzymes depend on the physical nature of surface groups of the solid phase. Changing the stability of enzymes in the system with solid phase is observed even the adsorption bond is less stable （in the case of DEAE- and CM-cellulose in acidic media）. Injection to the medium ethanol, surfactants, sodium chloride and changing the temperature of the incubation medium could prevent the negative effects of the solid phases. When sorsilen is used as solid phase, pepsin and acidic protease from Asp. awamori suffer from high surface inactivation. Various surfactants influence adsorption state of enzymes differently. Non-ionic surfactants （Triton X-100） prevent adsorption and restore catalytic properties of enzymes.

山梨醇脱氢酶性质研究

目的:山梨醇脱氢酶具有不完全氧化多种羟基化合物的特性,在工业中已广泛应用。该文研究该酶的酶学性质。方法:利用单因素实验对山梨醇脱氢酶的储藏稳定性和操作稳定性、反应液的pH和反应温度对酶活力的影响进行了研究,并进行了反应动力学研究。结果:反应液的pH值为5.5、反应温度为28℃时,酶活力最高;单批次反应过程中,酶基本没有失活。底物耗尽时,连续进行两次补加相同量的底物,酶活力有较小的降低。第一次重复时,酶活力有较小的降低;在第二次重复使用时,丧失40%的活力;山梨醇脱氢酶的底物特异性常数Ks为51 mmol/L,单位生物量的山梨醇脱氢酶最大活力Umax为1.7U/mg干细胞。结论:山梨醇脱氢酶的稳定性较高。

Variation in β-amylase activity and thermostability in Tibetan annual wild and cultivated barley genotypes

13-Amylase activity （BAA） and thermostability （BAT） are important traits for malt quality. In this study, 138 Tibetan annual wild barley accessions and 20 cultivated genotypes differing in BAh, were planted and analyzed in 2009 and 2012. Significant differences were detected among genotypes in BAA and BAT. The cultivated genotypes had a mean BAA of 1137.6 U/g and a range of from 602.1 to 1407.5 U/g, while the wild accessions had a mean of 1517.9 U/g and a range of from 829.7 to 2310.0 U/g. The cultivated genotypes had a mean relative residual 13-amylase activity （RRBAA） of 61.6% and a range of from 22.2% to 82.3%, while the wild barleys had a mean of 57.8% and a range of from 21.9% to 96.1%. Moreover, there was a significant difference among genotypes in the response of RRBAA to the temperature and duration of heat treatment. The wild barleys had wider variation in BAA and BAT than cultivated genotvDes.

Purification and characterization of CHpro1, a thermotolerant, alkali-stable and oxidation-resisting protease of Chumathang hotspring

Metagenomic approaches are recently used for searching novel open reading frames （ORFs） coding enzymes employed in pharmaceutical, rood industries, etc. In this study, a metagenornic library was constructed from Chumathang hotspring sediment DNA. The library consisted of approximately 9,000 clones and was screened for protease activity. A clone exhibiting protease activity was identified and named CHprol. Sequencing of CHprol revealed that the ORF encoded a functional protein of 363 amino acids belonging to peptidase S8-S53 superfamily. CHprol shared 41% sequence similarity with a reported protease （subtilase family） and 35 % structural similarity with the crystal structure of Pro-Tk sps. of Thermococcus kodarkaenasis. In silico modeling the 3D structure of CHprol showed that it has two beta sheets, 10 alpha helices and 11 strands. Catalytic triad prediction implied CHprol to be a serine protease. The optimum temperature and pH of the purified protease were found to be 80 ℃ and 11.0, respectively. The enzyme was active at 5 % concentration of hydrogen peroxide and retained 60 % of activity at 10 % concentration. The thermotolerant, alkalophilic and oxidant stable properties of the protease make it a potential can- didate for biotechnological applications.