Autohydrolysis of plant polysaccharides using transgenic hyperthermophilicenzymes

Commercial bioprocessing of plant carbohydrates, such as starch or cellulos e, necessitates the use of commodity enzyme additives to accelerate polysac charide hydrolysis. To simplify this procedure, transgenic plant tissues co nstitutively producing commodity enzymes were examined as a strategy for ac celerating carbohydrate bioprocessing. Hyperthermophilic glycosyl hydrolase s were selected to circumvent enzyme toxicity, because such enzymes are ina ctive at plant growth temperatures and are therefore physiologically benign . Transgenic tobacco lines were established that produced either a hyperthe rmophilic a-glucosidase or a P-glycosidase using genes derived from the arc haeon Sulfolobus solfataricus. Western blot and immunoprecipitation analyse s were used to demonstrate the presence of recombinant enzymes in plant tis sues. Transgenic enzyme levels exhibited an unusual delayed pattern of accu mulation while their activities survived plant tissue preservation. Transge nic plant protein extracts released glucose from purified polysaccharide su bstrates at appreciable rates during incubation in high-temperature reactio ns. Glucose was also produced following enzymatic treatment of plant extrac ts enriched for endogenous polysaccharides. Direct conversion of plant tiss ue into free sugar was evident using whole plant extracts of either transge nic line, and could be significantly accelerated in a synergistic manner by combining transgenic line extracts. (C) 2000 John Wiley & Sons, Inc.