Ghangas G S, Hu Y J, Wilson D B
Division of Biological Sciences, Cornell University, Ithaca, New York 14853.
J Bacteriol. 1989 Jun;171(6):2963-9. doi: 10.1128/jb.171.6.2963-2969.1989.
Thermomonospora fusca chromosomal DNA was partially digested with EcoRI to obtain 4- to 14-kilobase fragments, which were used to construct a library of recombinant phage by ligation with EcoRI arms of lambda gtWES. lambda B. A recombinant phage coding for xylanase activity which contained a 14-kilobase insert was identified. The xylanase gene was localized to a 2.1-kilobase SalI fragment of the EcoRI insert by subcloning onto pBR322 and derivatives of pBR322 that can also replicate in Streptomyces lividans. The xylanase activity produced by S. lividans transformants was 10- to 20-fold higher than that produced by Escherichia coli transformants but only one-fourth the level produced by induced T. fusca. A 30-kilodalton peptide with activity against both Remazol brilliant blue xylan and xylan was produced in S. lividans transformants that carried the 2.1-kilobase SalI fragment of T. fusca DNA and was not produced by control transformants. T. fusca cultures were found to contain a xylanase of a similar size that was induced by growth on xylan or Solka Floc. Antiserum directed against supernatant proteins isolated from a Solka Floc-grown T. fusca culture inhibited the xylanase activity of S. lividans transformants. The cloned T. fusca xylanase gene was expressed at about the same level in S. lividans grown in minimal medium containing either glucose, cellobiose, or xylan. The xylanase bound to and hydrolyzed insoluble xylan. The cloned xylanase appeared to be the same as the major protein in xylan-induced T. fusca culture supernatants, which also contained at least three additional minor proteins with xylanase activity and having apparent molecular masses of 43, 23, and 20 kilodaltons.
用EcoRI对嗜热栖热放线菌染色体DNA进行部分酶切，以获得4至14千碱基的片段，这些片段通过与λgtWES.λB的EcoRI臂连接用于构建重组噬菌体文库。鉴定出一个编码木聚糖酶活性的重组噬菌体，其含有一个14千碱基的插入片段。通过亚克隆到pBR322以及也能在变铅青链霉菌中复制的pBR322衍生物上，将木聚糖酶基因定位到EcoRI插入片段的一个2.1千碱基的SalI片段上。变铅青链霉菌转化子产生的木聚糖酶活性比大肠杆菌转化子产生的高10至20倍，但仅为诱导型嗜热栖热放线菌产生水平的四分之一。携带嗜热栖热放线菌DNA的2.1千碱基SalI片段的变铅青链霉菌转化子产生了一种对雷玛唑亮蓝木聚糖和木聚糖都有活性的30千道尔顿肽，而对照转化子未产生。发现嗜热栖热放线菌培养物中含有一种大小相似的木聚糖酶，该酶在以木聚糖或Solka Floc为生长底物时被诱导产生。针对从以Solka Floc培养的嗜热栖热放线菌培养物中分离的上清蛋白的抗血清抑制了变铅青链霉菌转化子的木聚糖酶活性。克隆的嗜热栖热放线菌木聚糖酶基因在含有葡萄糖、纤维二糖或木聚糖的基本培养基中生长的变铅青链霉菌中表达水平大致相同。该木聚糖酶与不溶性木聚糖结合并水解它。克隆的木聚糖酶似乎与木聚糖诱导的嗜热栖热放线菌培养上清液中的主要蛋白质相同，上清液中还含有至少另外三种具有木聚糖酶活性且表观分子量分别为43、23和20千道尔顿的次要蛋白质。
