Background: α-Amylases are starch-degrading enzymes and used widely, the study on thermostability of α-amylase is a central requirement for its application in life science and biotechnology. Objective: In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL. Methods: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (halflife, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template. Results: With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly higher than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α- helical contents increasing, and the transition from α-helices to β-sheet structures, which was obviously different in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure. Conclusion: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

The potential of using corn steep liquor powder (CSLP), peanut meal (PM), soybean meal (SM), cotton meal (CM) and urea as the substitute of yeast extract (YE) as the nitrogen source was investigated for producing succinic acid (SA). Actinobacillus succinogenes GXAS137 was used as the fermenting bacterium and sugarcane molasses was used as the main substrate. None of these materials were able to produce SA as high as YE did. The CSLP could still be considered as a feasible and inexpensive alternate for YE as the yield of SA produced using CSLP was second only to the yield of SA obtained by YE. The use of CSLP‐PM mixed formulation (CSLP to PM ratio = 2·6) as nitrogen source produced SA up to 59·2 g l⁻¹ with a productivity of 1·2 g l⁻¹ h⁻¹. A batch fermentation using a stirred bioreactor produced up to 60·7 g l⁻¹ of SA at the same formulation. Fed‐batch fermentation that minimized the substrate inhibition produced 64·7 g l⁻¹ SA. These results suggest that sugarcane molasses supplemented with a mixture of CSLP and PM as the nitrogen source could be used to produce SA more economically using A. succinogenes. Significance and Impact of the Study Succinic acid (SA) is commonly used as a platform chemical to produce a number of high value derivatives. Yeast extract (YE) is used as a nitrogen source to produce SA. The high cost of YE is currently the limiting factor for industrial production of SA. This study reports the use of a mixture of corn steep liquor powder (CSLP) and peanut meal (PM) as an inexpensive nitrogen source to substitute YE. The results showed that this CSLP‐PM mixed formulation can be used as an effective and economic nitrogen source for the production of SA.

Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of α2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction, the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases.