Mutations Asn20-->Cys/Ala27-->Cys (SS), Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Gly137-->Ala, Tyr312-->Trp and Ser436-->Pro in Aspergillus awamori glucoamylase, along with a mutation inserting a seven-residue loop between Tyr311 and Gly314 (311-314 Loop), were made to increase glucose yield from maltodextrin hydrolysis. No active Lys108-->Met glucoamylase was found in the supernatant after being expressed from yeast. Lys108-->Arg, 311-314 Loop and Tyr312-->Trp glucoamylases have lower activities than wild-type glucoamylase; other GAs have the same or higher activities. SS and 311-314 Loop glucoamylases give one-quarter to two-thirds the relative rates of isomaltose formation from glucose compared with glucose formation from maltodextrins at 35, 45 and 55 degrees C, correlating with up to 2% higher peak glucose yields from 30% (w/v) maltodextrin hydrolysis. Conversely, Lys108-->Arg glucoamylase has relative isomaltose formation rates three times higher and glucose yields up to 4% lower than wild-type glucoamylase. Gly137-->Ala and Tyr312-->Trp glucoamylases also give high glucose yields at higher temperatures. Mutated glucoamylases that catalyze high rates of isomaltose formation give higher glucose yields from shorter than from longer maltodextrins, opposite to normal experience with more efficient glucoamylases.

ABSTRACT. This study aimed to enhance the drug metabolism function of the human hepatoma cell line C3A and to explore the related significance for patients with severe liver disease. The important liver phase I and phase II drug metabolism enzymes, cytochrome P450 3A4 (CYP 3A4) and glutathione S-transferase A1 (GST A1), were constructed into a double expression vector and then transfected into C3A cells. Furthermore, in order to increase the expression of CYP 3A4 and GST A1, they were optimized according to human optimal codons. Another double-expression vector, pBudCE4.1-optimized CYP 3A4-optimized GST A1, was constructed and then transfected into C3A to establish a stable cell line. The drug metabolism function of C3A was evaluated. Sequence determination and analysis results showed that the recombinant plasmid pBudCE4.1-CYP 3A4-GST A1 met the application standard and its transfection was successful. The expression and activity of CYP 3A4 and GST A1 in unoptimized C3A cells were higher than those in blank C3A cells. Unoptimized C3A had a better drug metabolism function. Although some C3A cells transfected with pBudCE4.1-optimized CYP 3A4-optimized GST A1 survived, they grew slowly, and were therefore not applicable in clinical practice. Unoptimized C3A is superior to blank C3A in drug metabolism, and could be applied in the bioartificial liver support system as a new material.