Klug-Santner Barbara G, Schnitzhofer Wolfgang, Vrsanská Maria, Weber Jörg, Agrawal Pramod B, Nierstrasz Vincent A, Guebitz Georg M
Department of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
J Biotechnol. 2006 Feb 10;121(3):390-401. doi: 10.1016/j.jbiotec.2005.07.019. Epub 2005 Sep 15.
An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a molecular mass of 37.3+/-4.8 kDa as determined by SDS-PAGE and an isoelectric point of pH 8.5. Peptide mass mapping by nano-LC-MS of PL revealed 15% homology with a pectate lyase from Bacillus sp. The pectate lyase exhibited optimum activity at pH 8.5 and around 70 degrees C in Tris/HCl buffer. It showed a half-life at 30 degrees C of more than 75 h. Stability decreased with increasing temperature, extremely over 60 degrees C. The enzyme did not require Ca2+ ions for activity, and was strongly inhibited by EDTA and Co2+. PL was active on polygalacturonic acid and esterified pectin, but the affinity showed a maximum for intermediate esterified pectins and decreased over a value of 50% of esterification. The best substrate was 29.5% methylated pectin. PL cleaved polygalacturonic acid via a beta-elimination mechanism as shown by NMR analysis. PL released unsaturated tetragalacturonic acid from citrus pectin and polygalacturonic acid, but did not show any side activities on other hemicelluloses. On polygalacturonic acid PL showed a Km of 0.24 gl(-1) and a vmax of 0.72 gl(-1)min(-1). The applicability of pectate lyase for the bioscouring process was tested on a cotton fabric. Removal of up to 80% of pectin was proven by means of ruthenium red dyeing and HPAEC (65%). Structural contact angle measurements clearly indicated the increased hydrophilicity of enzyme treated fabrics.
基于胞外酶用于生物精练的潜力，分离出一种嗜碱细菌。该细菌被鉴定为短小芽孢杆菌BK2的一个新菌株，它能产生胞外内切果胶酸裂解酶PL（EC 4.2.2.2）。PL通过三步纯化至同质，经SDS-PAGE测定其分子量为37.3±4.8 kDa，等电点为pH 8.5。通过纳米液相色谱-质谱对PL进行肽质量图谱分析，结果显示其与芽孢杆菌属的一种果胶酸裂解酶有15%的同源性。该果胶酸裂解酶在Tris/HCl缓冲液中，pH 8.5和70℃左右时表现出最佳活性。它在30℃时的半衰期超过75小时。随着温度升高稳定性下降，在60℃以上时稳定性急剧下降。该酶的活性不需要Ca2+离子，且受到EDTA和Co2+的强烈抑制。PL对聚半乳糖醛酸和酯化果胶有活性，但对中等酯化度的果胶亲和力最大，当酯化度超过50%时亲和力下降。最佳底物是29.5%甲基化的果胶。如NMR分析所示，PL通过β-消除机制裂解聚半乳糖醛酸。PL从柑橘果胶和聚半乳糖醛酸中释放出不饱和四聚半乳糖醛酸，但对其他半纤维素没有任何副活性。在聚半乳糖醛酸上，PL的Km为0.24 g l-1，vmax为0.72 g l-1 min-1。在棉织物上测试了果胶酸裂解酶在生物精练过程中的适用性。通过钌红染色和高效阴离子交换色谱法（HPAEC）证明，果胶去除率高达80%（HPAEC法为65%）。结构接触角测量清楚地表明，酶处理后的织物亲水性增加。
