Construction and Characterization of a Novel Bacmid AcBac-Syn Based on a Synthesized Baculovirus Genome

Virologica Sinica - Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides. Homologous recombination and Bac-to-Bac system have been the...     

定点突变提高木聚糖酶Umxyn10A的热稳定性

木聚糖酶是微生物半纤维素降解体系中的一种关键酶,被广泛地应用在工业中的多个领域。本研究为了提高木聚糖酶Umxyn10A (ABL73-883.1)的热稳定性,将Umxyn10A与GH 10家族4种耐热木聚糖酶进行多序列同源比对以及三维结构的同源建模分析,选定了Umxyn10A第31位氨基酸位点进行定点突变,将氨基酸Ala (A)突变为Phe (F)。分别将Umxyn10A和Umxyn10AA31F在大肠杆菌中进行重组表达,分析2种重组酶的酶学特性,结果发现,Umxyn10AA31F的最适反应温度为85℃,较野生重组酶提高了5℃;在65℃下的半衰期为105 min,较野生重组酶(15 min)提高了6倍;在70℃下突变重组酶的半衰期为15 min,较野生重组酶(5 min)提高了2倍;与此同时突变重组酶的pH耐受区间较原酶也有一定的增大。结果表明,将第31位氨基酸位点Ala突变为Phe能够显著的提高Umxyn10A的热稳定性。     

Mechanism of regulating the expression of λN gene by ribosomal protein at translational level

In ribosomal protein S12 mutant or L24 mutant the expression of λN gene was depressed at translational level. To study its mechanism the λN gene region of λN -lacZ gene fusion was trimmed from its 5′ end to 3′ end with DNA exonuclease III (DNA cxoIII) in order to alter the TIR (translational initiation region) and the ding region of λN gene. After DNA sequencing 23 species of different λN-lacZ fused genes were obtained. The β-galactosidase activities of these deletants in ribosomal protein mutant were compared with that in wild type strain. The result indicated that (i) S12 mutant could affect 305 subunit’s binding to the TIR of λN gene messenger and cause the difficulty in forming 30s initiation complex and then decrease the efficiency of translational initiation; (ii) in S12 mutant the coding region of λN gene alw affected the expression λN gene; (iii) in L24 mutant the inhibition of λN gene expression was not related to translational initiation and the 5′ end of the coding region of λN gene, but related to the 3′ end of λN gene.