OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT BARLEY ALPHA-AMYLASE-1 AND ALPHA-AMYLASE-2 SECRETED BY THE METHYLOTROPHICYEAST PICHIA-PASTORIS

Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pic hia pastoris at up to 50 and 1 mg/liter, respectively, representing ap proximately a 50-fold increase compared to the levels of the heterolog ous expression by Saccharomyces cerevisiae. The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL- D2. Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in qu antitative yield by affinity chromatography on beta-cyclodextrin-Sepha rose. The N-terminal sequence, pi, and M(r) indicated that native-like processing took place, Electrospray ionization mass spectrometry, how ever, revealed microheterogeneity for recombinant isozyme 1. While M(r ) of one recombinant isozyme 1 form of 45,452 was in excellent agreeme nt with a value of 45,447 calculated from the sequence, liquid chromat ography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectr ometry/mass spectrometry system identified additional recombinant isoz yme 1 forms to be glycosylated on Thr(410), N-acetylated on His(1), S- glutathionylated on Cys(95), or C-terminally truncated of -(412)RS, (4 11)QRS, and -(410)LQRS. The recombinant enzymes and the cu-amylases fr om barley malt closely resembled each other in enzymatic activity on i nsoluble Blue Starch, amylose of degree of polymerization 17, and 2-ch loro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependenc y of activity, Pichia pastoris thus produced in high yields recombinan t cu-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts. This greatly facilitates futu re mutational analysis of barley alpha-amylase in order to probe struc ture/function relationships. (C) 1996 Academic Press, Inc.