Over the past decades, nitrilases have drawn considerable attention because of their application in nitrile degradation as prominent biocatalysts. Nitrilases are derived from bacteria, filamentous fungi, yeasts, and plants. In-depth investigations on their natural sources function mechanisms, enzyme structure, screening pathways, and biocatalytic properties have been conducted. Moreover, the immobilization, purification, gene cloning and modifications of nitrilase have been dwelt upon. Some nitrilases are used commercially as biofactories for carboxylic acids production, waste treatment, and surface modification. This critical review summarizes the current status of nitrilase research, and discusses a number of challenges and significant attempts in its further development. Nitrilase is a significant and promising biocatalyst for catalytic applications.

MicroRNAs are small, highly conserved, non‐coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition, and are widely involved in carcinogenesis and cancer development. In this study, the expression profile of microRNA‐133a (miR‐133a) was examined in breast cancer cells and breast cancer tissues. The results showed that expression of miR‐133a in both breast cancer cells and breast cancer tissues was significantly down‐regulated. Over‐expression of miR‐133a in tumor cells arrested the cell cycle by drastically decreasing the G₂/S phase and retarded the newly synthesized DNA, suggesting a regulatory role for miR‐133a in proliferation of breast cancer cells. Bioinformatics prediction showed that epidermal growth factor receptor (EGFR) is a potential target for miR‐133a. A dual luciferase reporter gene assay showed that miR‐133a bound to the 3′ UTR of EGFR but not a mutated 3′ UTR, thereby down‐regulating the protein expression level. Accordingly, we found that expression of EGFR protein decreased with increased expression of miR‐133a in MCF‐7 and MDA‐MB‐231 cells. Over‐expression of miR‐133a in breast cancer cells resulted in suppression of the level of phosphorylated Akt protein (p‐Akt) and inhibition of p‐Akt nuclear translocation. These results demonstrate that miR‐133a, which may act as a tumor suppressor in breast cancer, regulates the cell cycle and proliferation in tumorigenesis by targeting EGFR through the downstream signal molecule Akt. Overall, these results show that miR‐133a may be used as biomarker and/or therapeutic target for diagnosis and therapy of breast cancer.

Bacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA). The promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the −10 and −35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level. The auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression system that has an expression pattern that is split between cell-growth and over-expression, leading to an increase in cell density and elevating the overall expression levels of heterologously expressed proteins. The broad applicability of this system and inducer-free expression property in B. subtilis facilitate the industrial scale-up and medical applications for the over-production of a variety of desired proteins.

Bacillus subtilis, a Gram-positive organism, has been developed to be an attractive expression platform to produce both secreted and cytoplasmic proteins owing to its prominent biological characteristics. We previously developed an auto-inducible expression system containing the srfA promoter (P srfA ) which was activated by the signal molecules acting in the quorum-sensing pathway for competence. The P srfA promoter exhibited the unique property of inducer-free activity that is closely correlated with cell density. To improve the P srfA -mediated expression system to the high-cell-density fermentation for industrial production in the B. subtilis mutant strain that is unable to sporulate, a spore mutant strain BSG1682 was developed, and the P srfA promoter was enhanced by promoter engineering. Using green fluorescent protein (GFP) as the reporter, higher fluorescent intensity was observed in BSG1682 with expression from either plasmid or chromosome than that of the wild type B. subtilis 168. Thereafter, the P srfA was engineered, yielding a library of P srfA derivatives varied in the strength of GFP expression. The P23 promoter exhibited the best performance, almost twofold stronger than that of P srfA . Two heterologous proteins, aminopeptidase (AP) and nattokinase (NK), were successfully overproduced under the control of P23 in BSG1682. Finally, the capacity of the expression system was demonstrated in batch fermentation in a 5-L fermenter. The expression system demonstrates prominence in the activity of the auto-inducible promoter. Desired proteins could be highly and stably produced by integrating the corresponding genes downstream of the promoter on the plasmid or the chromosome in strain BSG1682. The expression system is conducive to the industrial production of pharmaceuticals and heterologous proteins in high-cell-density fermentation in BSG1682.