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Objective: To study enzymatic properties of intracellular maltogenic amylase (BMAL) in Bacillus sp. B110, purification, gene cloning, heterologous expression and purification were performed. It laid a foundation for later research on the development of new starch processing enzymes. Method: The full-length of BMAL gene was amplified by PCR and over-expressed and heterologous expressed in Escherichia coli. The recombinant enzyme was purified with nickel affinity chromatography (Ni2+-NTA), and its enzymatic characteristic were determined. The amino acid sequence of BMAL was analyzed by sequence analysis tools BioEdit and MEGA tools, and the three-dimensional model was predicted by alpha fold2. Results: The nucleotide sequence analysis revealed an open reading frame (ORF) of 1770 bp encoded a putative protein of 589 aa residues. The recombinant enzyme was purified with nickel affinity chromatography (Ni2+-NTA). SDS-PAGE analysis of the purified enzyme revealed that the molecular mass of BMAL was 63 kDa. The primary structure of BMAL was similar to those of MAases from B. subtilis 168 and B. subtilis SUH4-2, such as possession of an extra domain at its N-terminal and had a catalytic triad Asp328-Glu357-Asp424. The purified recombinant enzyme rBMAL had an optimum temperature of 45 ℃ and an optimum pH around 6.0. The rBMAL retained about 60% activity after 7 h of incubation at below 30 ℃, but it lost 98% of the original activity after 2 h of incubation at 60 ℃. These results revealed that the rBMAL was inthermostability. The activity of rBMAL was stable in pH7.0~9.5 at 4 ℃ for 12 h. In the presence of 1 mmol/L metal ion Mg2+, the activity of rBMAL increased by 36%, while 1 mmol/L Ni2+, Fe3+, Co2+, Cu2+, Zn2+, Al3+, Ca2+ inhibited the activity of rBMAL, which decreased by 85%~48%. The recombinant enzyme activity was inhibited by methanol, acetone, ethanol, acetonitrile, EDTA and SDS, and the enzyme activity was decreased to 32.3%~64.8%. Conclusion: The intracellular maltose amylase BMAL from Bacillus sp. B110 had high catalytic capacity and pH stability, which had potential application in bread baking industry.

Disregulation of genes making up the mammalian circadian clock has been associated with different forms of cancer. This study aimed to address how the circadian clock genes behave over the course of treatment for both the acute and chronic forms of leukemia and whether any could be used as potential biomarkers as a read-out for therapeutic efficacy. Expression profiling for both core and ancillary clock genes revealed that the majority of clock genes are down-regulated in acute myeloid leukemia patients, except for Cry2, which is up-regulated towards the end of treatment. A similar process was seen in acute lymphocytic leukemia patients; however, here, Cry2 expression came back up towards control levels upon treatment completion. In addition, all of the core clock genes were down-regulated in both chronic forms of leukemia (chronic myeloid leukemia and chronic lymphocytic leukemia), except for Cry2, which was not affected when the disease was diagnosed. Furthermore, the NAD(+) – dependent protein deacetylase Sirt1 has been proposed to have a dual role in both control of circadian clock circuitry and promotion of cell survival by inhibiting apoptotic pathways in cancer. We used a pharmacological-based approach to see whether Sirt1 played a role in regulating the circadian clock circuitry in both acute and chronic forms of leukemia. Our results suggest that interfering with Sirt1 leads to a partial restoration of BMAL1 oscillation in chronic myeloid leukemia patient samples. Furthermore, interfering with Sirt1 activity led to both the induction and repression of circadian clock genes in both acute and chronic forms of leukemia, which makes it a potential therapeutic target to either augment existing therapies for chronic leukemia or to act as a means of facilitating chronotherapy in order to maximize both the effectiveness of existing therapies and to minimize therapy-associated toxicity.