Xiaoyan Xiong, William S. Adney, Todd B. Vinzant, Yat-Chen Chou,
Michael E. Himmel1, and Steven R. Thomas

Biotechnology Center for Fuels and Chemicals, National Renewable
Energy Laboratory, 1617 Cole Boulevard, Golden, CO 80401

Active, thermostable Microbispora bispora Bgl В (i-D-glucosidase was expressed in Streptomyces lividans TK24 cells transformed with plasmid pIJ702 carrying the bglB coding sequence under the control of Streptomyces longisporus STI-II trypsin inhibitor promoter. The recombinant enzyme, 5/Bgl B, has a molecular weight of 54 kDa, an isoelectric pH of 5.0, shows resistance to glucose inhibition, and is optimally active on o-nitrophenyl р-D-glucopyranoside at 57°C. This recombinant (i-D-glucosidase was more active on aryl-glycosides than on cellobiose. We also report a successful mutagenesis strategy used to achieve increased levels of 5/Bgl В expression in this host organism. Screening mutants created by low fidelity PCR using Taq polymerase in the presence of manganese ion revealed a series of up-regulated clones, one yielding 235 mg/L of 5/Bgl B.

p-D-glucoside glucohydrolases (EC 3.2.1.21), or p-D-glucosidases, catalyze the hydrolysis of O-glycosyl bonds in aryl — and alkyl-glucosides, as well as in many p-linked disaccharides and some oligosaccharides. These enzymes are produced by plants, animals, and most known microbiota and have been extensively reviewed (7,2). Each of these enzymes displays a distinct pattern of relative activity on an array of p-glucosides. A subset of p-D — glucosidases are especially proficient at the hydrolysis of cellobiose, and are often referred to as cellobiases.

In general, thermotolerant enzymes have higher turnover rates and better tolerate the stresses of use in large-scale processes. Thus, they are of interest to industry (5). Thermophilic P-D-glucosidases have been found in Clostridium stercorarium, T^ = 65°C

(4) ; Clostridium thermocellum NQB 10682, T^ = 65°C (5); Thermomonospora sp. strain YX, T^ = 55°C (6); and Acidothermus cellulolyticus, T^ = 75°C (7). Caldophilic P-D — glucosidases have also been isolated from Caldocellum saccharolyticum, Topt = 85°C (5);

Corresponding author

© 1997 American Chemical Society

Clostridium thermocopriae JT3-3, = 80°C (9); and Thermus strain Z-l, Topt = 85°C

m.

Typically, native strains are not prolific producers of p-D-glucosidase activity, so researchers have used recombinant DNA expression strategies to obtain reasonable quantities of these enzymes. Genetically engineered expression from E. coli of various thermostable P-D-glucosidases including C. saccharolyticum (5), C. thermocellum NUB 10682 (77), C. thermocellum NOB 10682 (72), and Rhodothermus marinus (13) has been demonstrated in several laboratories using lambda phage or plasmid vectors in E. coli and other host bacteria.

In 1986, Waldron and со workers reported the isolation of a new thermo tolerant actinomycete, Microbispora bispora, from warm compost (14). This microorganism produced thermostable p-D-glucosidase activity that was resistant to inhibition by glucose concentrations as high as 30% w/v. Upon screening a genomic library of M. bispora DNA, Wright (75) discovered two DNA fragments of 4.0 kb and 2.1 kb coding for two distinct p-D-glucosidases, Bgl A and Bgl B, respectively. The bglB gene encodes M. bispora p-D-glucosidase B, MBgl B, which is highly resistant to feedback inhibition by glucose, but is expressed only very weakly from its native promoter in E. coli. We report the cloning of the coding sequence for MBgl В under the control of the STI-II trypsin inhibitor promoter from Streptomyces longisporus (16) in plasmid pIJ702, as well as the expression and characterization of active, thermostable enzyme from transformed Streptomyces lividans TK24, S/Bgl B. We also report on our efforts to mutagenize the STI-II promoter to achieve higher levels of 5/Bgl В expression in this host organism.