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Bacillus subtilis endo-beta-1,4-glucanase (Cel5A) hydrolyzes cellulose by cleavage of the internal bonds in the glucose chains, producing new ends randomly. Using directed evolution techniques of error-prone polymerase chain reaction (PCR) and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68-fold increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78, and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme. Moreover, V74A and D272G of M44-11, which were not located in the substrate binding sites and the catalytic center, might result in improved stability and catalytic activity. These results provided useful references for directed evolution of the enzymes that belonged to the glycoside hydrolase family 5 (GH5).

ORCID IDs: 0000-0003-0210-9408 (Z.Z.); 0000-0002-6839-3272 (X.H.); 0000-0001-5113-7750 (J.W.); 0000-0002-5098-8681 (J.D.).Cold acclimation is an important process by which plants respond to low temperature and enhance their winter hardiness. C-REPEAT BINDING FACTOR1 (CBF1), CBF2, and CBF3 genes were shown previously to participate in cold acclimation in Medicago truncatula. In addition, MtCBF4 is transcriptionally induced by salt, drought, and cold stresses. We show here that MtCBF4, shown previously to enhance drought and salt tolerance, also positively regulates cold acclimation and freezing tolerance. To identify molecular factors acting upstream and downstream of the MtCBF4 transcription factor (TF) in cold responses, we first identified genes that are differentially regulated upon MtCBF4 overexpression using RNAseq Digital Gene Expression Profiling. Among these, we showed that MtCBF4 directly activates the transcription of the COLD ACCLIMATION SPECIFIC15 (MtCAS15) gene. To gain insights into how MtCBF4 is transcriptionally regulated in response to cold, an R2R3-MYB TF, MtMYB3, was identified based on a yeast one-hybrid screen as binding directly to MYB cis-elements in the MtCBF4 promoter, leading to the inhibition of MtCBF4 expression. In addition, another MYB TF, MtMYB61, identified as an interactor of MtMYB3, can relieve the inhibitory effect of MtMYB3 on MtCBF4 transcription. This study, therefore, supports a model describing how MtCBF4 is regulated by antagonistic MtMYB3/MtMYB61 TFs, leading to the up-regulation of downstream targets such as MtCAS15 acting in cold acclimation in M. truncatula.