杂色云芝漆酶的分离、纯化和酶学特性研究

杂色云芝发酵液,经超滤、DEAE-Sephadex A250柱层析两步分离、纯化出两型漆酶。以愈创木酚与酶带在适当条件下反应呈棕红色谱带鉴定出漆酶谱带。两型漆酶分别命名为LacA和LacB。经测定两漆酶亚基的分子量分别为66.0kDa和30.7kDa;等电点pI分别为4.79和5.18;LacA的最适作用温度是40℃,LacB为50℃;保温2h, LacA的半失活温度为60℃,LacB为90℃;LacA和LacB催化愈创木酚的酶活最适pH值均为4.0; 二者的稳定pH值也都在2.5~5.5之间。LacA催化氧化愈创木酚的Km和Vmax分别为286.5molL-1和292.4nmol mL-1 min-1;LacB催化氧化愈创木酚的Km和Vmax分别为813.0mol L-1和127.4nmol mL-1 min-1。     

Oil and fat hydrolysis with lipase fromAspergillus sp.

Hydrolysis of olive oil, soybean oil, mink fat, lard, palm oil, coconut oil, and a hydrogenated, hardened oil with lipase from anAspergillus sp. has been studied. The lipase had high specific activity (60,000 U/g) and did not show any positional specificity. The lipase proved to be a more effective catalyst than Lipolase fromA. oryzae, with an optimal activity at 37°C and pH 6.5–7.0. It was activated by Ca²⁺ but inactivated by organic solvents such as isopropanol and propanone. All substrates examined could be hydrolyzed to corresponding fatty acids with this enzyme at concentrations of 5–30 U/meq with yields of 90–99% in 2–24 h. The degree of hydrolysis was almost logarithmically linear with reaction time and occurred in two stages. The lipase was stable and could be repeatedly recycled for hydrolysis.     

Inhibitory effect of Cr（Ⅵ） on activities of soil enzymes

To evaluate the influence of various Cr(VI) concentrations (0.05, 0.25, 0.50, 1.00 and 2.00 g/kg) on the activity of soil enzymes, the activities of catalase, polyphenol oxidase, dehydrogenase, alkaline phosphatase in soils were investigated in the incubation experiment with a period of 35 d. The results indicate that all the tested Cr(VI) concentrations significantly inhibit dehydrogenase activity by over 70% after 35 d. The activity of alkaline phosphatase is slightly inhibited during the whole experiment except for on the day 7. Cr(VI) has no obvious effect on the activity of catalase in soil. On the contrary, Cr(VI) stimulates the activity of polyphenol oxidase. The results suggest that dehydrogenase activity can be used as an indicator for assessing the severity of chromium pollution. Foundation item: Projects(2006AA06Z374, 2007AA021304) supported by the National High-Tech Research and Development Program of China; Project(2008SK2007) supported by the Key Program of Science and Technology of Hunan Province, China     

金属离子对红球菌腈水合酶活力的影响

不同金属离子对红球菌（Rhodococcus sp．）TCCC 28001的生长及其腈水合酶的酶活有着不同程度的影响．菌株TCCC 28001产生的腈水合酶是钴型,且Co^2＋对该腈水合酶诱导激活的最适浓度为10×10^-5mol／L．选择酵母粉中含有且含量波动较大的5种金属离子,考察了在添加10×10^-5 mol／L Co^2＋诱导激活下,5种金属离子对酶活的影响．结果表明：Fe^2＋,Mn^2＋,Mo^6＋,Cu^2＋4种金属离子具有促进作用,Zn^2＋产生轻微抑制作用．6×10^-5mol／L Fe^2＋的促进效果显著,使菌株TCCC 28001的酶活从453U／mL增加到1941U／mL,提高了328％．     

采前水杨酸处理对哈密瓜果实防御酶的诱导

采用1.0、2.0、3.0 mmol/L水杨酸分别在"蜜农9号"哈密瓜开花前1周、幼果期(花后2周)、果实迅速膨大期(花后3周)和网纹形成期(花后4周)对植株进行4次喷洒.依次在幼果期、膨大期、网纹形成期和成熟期进行取样.分析不同浓度水杨酸处理后果实体内3种防御性酶活性的变化.结果表明:采前1.0、2.0、3.0 mmol/L水杨酸处理均能有效地提高哈密瓜果实中过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)活性,尤其以2.0 mmol/L水杨酸处理酶活性增加最为显著.     

Fractionation of transgenic corn for recovery of recombinant enzymes

Two germ-separation methods, dry-milling and density separation by flotation, were evaluated for recovering recombinant β-glucuronidase (rGUS) that accumulated primarily in the germ of transgenic corn. The dry-milling process consisted of (i) seed tempering, (ii) degerming with a horizontal-drum degermer/dehuller, (iii) particle size fractionation with standard sieves, (iv) germ and endosperm separation by roller milling and sifting, and (v) removal of hulls by aspiration. Sieves nos. 5, 6, and 7 retained the majority of germ, and subfractions from these sieves were pooled as a germ-rich fraction. Mass balances showed that the germ-rich fraction, which constituted 17% of the total dry-milled corn weight, contained 49% of rGUS activity and 64% of the total recoverable oil. Germ fractionation by flotation was tested as a proof-of-concept method aimed at separating corn fractions based on their difference in specific gravity (sp gr). The process consisted of impact-grinding of corn kernels followed by density separation using 1.15 or 1.3 specific gravity sodium nitrate solution. The oil-containing germ fraction floated, whereas the heavier endosperm fraction sedimented. The flotation method was simpler and resulted in higher enzyme recovery, that is, the germ-rich fraction was 20% (w/w) of the initial corn weight, and accounted for 80% of rGUS activity and 77% of total oil. The sodium nitrate solution did not have an adverse effect on the enzyme activity.     

Activity and stability ofPenicillium cyclopium lipase in surfactant and detergent solutions

Activity and stability of an alkaline lipase fromPenicillium cyclopium var.album (PG 37) were studied in surfactant and detergent solutions. Three anionic surfactants [Na salts of C₁₂SO₄ ^? (sodium dodecyl sulfate), C₁₂ØSO₃/^? (linear alkyl benzene sulfonate), and C₁₁COO^? (laurate)] and four homologous series of nonionic surfactants of C_12–15 polyoxethylenated fatty alcohols (AEO₃, AEO₅, AEO₇, and AEO₉) were evaluated. At a concentration range of 3.2–40 μM, sodium dodecyl sulfate and laurate stimulated the activity of PG 37 lipase. At concentrations greater than 5.6 μM, linear alkylbenzene sulfonate inhibited PG 37 lipase activity. Nonionic surfactants, AEO₅ and AEO₇, in the concentration range of 0.25–20 mM, enhanced and stabilized the activity of PG 37 lipase. The presence of PG 37 lipase in detergent formulaton improved detergency ～20%. The mechanism of inhibition of the lipolytic activity of PG 37 lipase is proposed to be partly due to the formation of inactive (BR)_n-E complex between the hydrophobic moiety of the surfactants and the surface of the lipase. Conversely, formation of a soluble (RB)_n-E complex between the hydrophilic group of the surfactant and lipase may account for the increased lipolytic activity of PG 37 lipase.     

关于木素生物降解酶类活力测定问题的讨论

由于木素结构和生物降解过程复杂多样，全面测定一个菌株的木素降解活力也比较困难，目前已发现三类主要参与木素降解的酶类（LIP、MnP、漆酶），但至今很少有国际上公认的酶活力测定方法，也未见到能把某一菌株木素降解酶类的活力与其在木质纤维材料中降解木素的能力定量关联的报导。本文综合评述了几类主要的木素降解酶类活力测定方法的作用机制和存在的问题，指出一个菌株的木素降解活力是由它的生理特性和总体代谢能力共同决定的，仅由一类或几类酶活力的表现水平难以对其作出确切的评价，从这个角度出发进行研究才能更客现他认识菌株的木素降解活力．     

两种纤维素酶水解漂白针叶木纤维能力的比较

采用复合纤维素酶Celluclast 1.5L和内切纤维素酶Novozym 476等分别对漂白针叶木纤维进行了水解实验.通过对比两种酶的酶活力大小以及水解后纤维得率的变化情况,分析比较了两种纤维素酶水解纤维素纤维的能力大小.其结果显示:纤维素酶Celluclast 1.5L具有较高的滤纸酶活,而纤维素酶Novozym 476具有较高的CMC酶活.经纤维素酶Celluclast 1.5L水解处理后,随着酶用量的增加或酶处理时间的延长,纤维得率急速下降,当酶用量为20.0FPU/g、水解时间为2h时,纤维得率约为81%;当酶用量为10.0FPU/g、水解时间为48h时,纤维得率仅为55.34%,这说明纤维素酶Celluclast 1.5L对纤维素具有很强的水解作用,它不仅可以作用于纤维素的无定形区,同时也可以作用于纤维素的结晶区.纤维素酶Novozym 476亦能使纤维素发生一定程度的水解,但催化水解能力远低于纤维素酶Celluclast 1.5L,当Novozym 476用量为50.0CMCU/g时,仅有5%左右的纤维素水解成为葡萄糖.     

人工代谢算法在故障诊断中的应用

从生物体新陈代谢的生理机能出发,建立了基于酶催化的人工代谢算法模型．通过对代谢算子功能的 分析提出了模式定理的概念,并证明了算法的收敛性．利用酶与底物相匹配的原理,通过代谢操作找出酶与底物的 最优匹配,从而找出对应故障现象的原因．在寻优过程中,不断地通过凋亡算子将不可能成立的原因筛除,降低了 故障诊断的成本．实例分析表明,该算法能有效地进行故障定位,尽可能地缩小了不必要的搜索空间．