Abstract
The cellulase producing ascomycete, Trichoderma reesei (Hypocrea jecorina), is known to secrete a range of enzymes important for ethanol production from lignocellulosic biomass. It is also widely used for the commercial scale production of industrial enzymes because of its ability to produce high titers of heterologous proteins. During the secretion process, a number of post-translational events can occur, however, that impact protein function and stability. Another ascomycete, Aspergillus niger var. awamori, is also known to produce large quantities of heterologous proteins for industry. In this study, T. reesei Cel7A, a cellobiohydrolase, was expressed in A. niger var. awamori and subjected to detailed biophysical characterization. The purified recombinant enzyme contains six times the amount of N-linked glycan than the enzyme purified from a commercial T. reesei enzyme preparation. The activities of the two enzyme forms were compared using bacterial (microcrystalline) and phosphoric acid swollen (amorphous) cellulose as substrates. This comparison suggested that the increased level of N-glycosylation of the recombinant Cel7A (rCel7A) resulted in reduced activity and increased non-productive binding on cellulose. When treated with the N-glycosidase PNGaseF, the molecular weight of the recombinant enzyme approached that of the commercial enzyme and the activity on cellulose was improved.
Highlights
It is estimated that more than a billion tons of lignocellulosic plant biomass could be utilized each year to produce liquid biofuels in North America alone [1]
SDS-PAGE analysis The recombinant Cel7A (rCel7A) has a higher apparent molecular weight than Trichoderma expressed Cel7A when analyzed by SDSPAGE (Figure 1)
The rCel7A band appears broader and more diffuse which is typical of protein with polydispersity of molecular weight due to glycosylation (Figure 1)
Summary
It is estimated that more than a billion tons of lignocellulosic plant biomass could be utilized each year to produce liquid biofuels in North America alone [1]. A major focus of research in the biofuels industry has been to address issues of enzyme production costs and performance. Developing low-cost enzymes remains a priority for both the Department of Energy's Office of the Biomass Program and for private industry. To achieve these goals, scientific advances in enzyme technologies, including enzyme production and performance, are required. Tactics proposed to solve the biomass conversion cost goals are diverse and include in planta enzyme expression, consolidated bioprocessing, enzymes engineered by rational design, enzymes improved by directed evolution, the development of hyper-producing fungal hosts, and many (page number not for citation purposes). In each case, understanding those factors that impact the heterologous and/or hyper-expression of enzymes is critical for success
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