Kubicek C P, Messner R, Gruber F, Mandels M, Kubicek-Pranz E M
Institut für Biochemische Technologie und Mikrobiologie, Abteilung für Mikrobielle Biochemie, TU Wien, Austria.
J Biol Chem. 1993 Sep 15;268(26):19364-8.
We prepared [U-14C]cellobiose by cultivating Acetobacter pasteurianus in the presence of [U-14C]glucose and hydrolyzing the [U-14C]cellulose formed with beta-glucosidase-free cellulase from Trichoderma reesei. This 14C-labeled cellobiose was used to investigate the presence of an uptake system for cellobiose in T. reesei. Evidence was obtained for the presence of a high affinity (Km for cellobiose 0.3 microM) but low activity (2.5 milliunits/mg fungal dry weight) cellobiose permease. The permease is formed constitutively, but higher levels are formed after addition of sophorose (glucosyl-beta-1,2-diglucoside), a reputed cellulase inducer. The permease appears to be specific for beta-diglucosides, as the uptake of [U-14C]cellobiose is inhibited by sophorose, gentiobiose (glucosyl-beta-1,3-glucoside), and cellobiose. Under these conditions, cellooligodextrines (n, 4-7; final concentration, 1 mM) are not inhibitors. Glucose, but no other monosaccharides, inhibits the permease. The hypersecretory mutant T. reesei RUT C-30 exhibits elevated permease activities, whereas in T. reesei QM 9979, a mutant strain defective in the induction of cellulases by cellulose or sophorose, strongly reduced permease activities were demonstrated. The results stress a hitherto not recognized point of control in the induction of cellulases by T. reesei at the level of uptake of cellulose oligosaccharides.
我们通过在[U-14C]葡萄糖存在的条件下培养巴氏醋杆菌,并使用里氏木霉不含β-葡萄糖苷酶的纤维素酶水解形成的[U-14C]纤维素来制备[U-14C]纤维二糖。这种14C标记的纤维二糖被用于研究里氏木霉中纤维二糖摄取系统的存在情况。已获得证据表明存在一种高亲和力(纤维二糖的Km为0.3 microM)但低活性(2.5毫单位/毫克真菌干重)的纤维二糖通透酶。该通透酶组成性形成,但在添加槐糖(葡萄糖基-β-1,2-二葡萄糖苷)(一种著名的纤维素酶诱导剂)后形成水平更高。该通透酶似乎对β-二糖苷具有特异性,因为[U-14C]纤维二糖的摄取受到槐糖、龙胆二糖(葡萄糖基-β-1,3-葡萄糖苷)和纤维二糖的抑制。在这些条件下,纤维寡糖(n,4 - 7;终浓度,1 mM)不是抑制剂。葡萄糖而非其他单糖会抑制该通透酶。超分泌突变体里氏木霉RUT C - 30表现出升高的通透酶活性,而在里氏木霉QM 9979(一种在纤维素或槐糖诱导纤维素酶方面存在缺陷的突变菌株)中,通透酶活性被证明大幅降低。这些结果强调了里氏木霉在纤维素寡糖摄取水平上诱导纤维素酶过程中一个迄今未被认识到的控制点。