scholarly journals Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Junxian Zheng ◽  
Taowei Yang ◽  
Junping Zhou ◽  
Meijuan Xu ◽  
Xian Zhang ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Enzyme Inactivation ◽  
Cysteine Residues ◽  
Candida Boidinii ◽  
Wild Type ◽  
Nadh Regeneration ◽  
Content Type ◽  
The Cost

ABSTRACT NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10C fdh ) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10C fdh , which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances. IMPORTANCE FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.

scholarly journals Avoidance of Autophagy Mediated by PlcA or ActA Is Required for Listeria monocytogenes Growth in Macrophages

2015 ◽  
Vol 83 (5) ◽  
pp. 2175-2184 ◽  
Author(s):  
Gabriel Mitchell ◽  
Liang Ge ◽  
Qiongying Huang ◽  
Chen Chen ◽  
Sara Kianian ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Wild Type Strain ◽  
Fold Increase ◽  
Host Cells ◽  
Listeriolysin O ◽  
Wild Type ◽  
Conjugation System ◽  
Content Type ◽  
A Cell ◽  
Gain Access

Listeria monocytogenesis a facultative intracellular pathogen that escapes from phagosomes and grows in the cytosol of infected host cells. Most of the determinants that govern its intracellular life cycle are controlled by the transcription factor PrfA, including the pore-forming cytolysin listeriolysin O (LLO), two phospholipases C (PlcA and PlcB), and ActA. We constructed a strain that lacked PrfA but expressed LLO from a PrfA-independent promoter, thereby allowing the bacteria to gain access to the host cytosol. This strain did not grow efficiently in wild-type macrophages but grew normally in macrophages that lacked ATG5, a component of the autophagy LC3 conjugation system. This strain colocalized more with the autophagy marker LC3 (42% ± 7%) at 2 h postinfection, which constituted a 5-fold increase over the colocalization exhibited by the wild-type strain (8% ± 6%). While mutants lacking the PrfA-dependent virulence factor PlcA, PlcB, or ActA grew normally, a double mutant lacking both PlcA and ActA failed to grow in wild-type macrophages and colocalized more with LC3 (38% ± 5%). Coexpression of LLO and PlcA in a PrfA-negative strain was sufficient to restore intracellular growth and decrease the colocalization of the bacteria with LC3. In a cell-free assay, purified PlcA protein blocked LC3 lipidation, a key step in early autophagosome biogenesis, presumably by preventing the formation of phosphatidylinositol 3-phosphate (PI3P). The results of this study showed that avoidance of autophagy byL. monocytogenesprimarily involves PlcA and ActA and that either one of these factors must be present forL. monocytogenesgrowth in macrophages.

scholarly journals Substitutions at Position 105 in SHV Family β-Lactamases Decrease Catalytic Efficiency and Cause Inhibitor Resistance

2012 ◽  
Vol 56 (11) ◽  
pp. 5678-5686 ◽  
Author(s):  
Mei Li ◽  
Benjamin C. Conklin ◽  
Magdalena A. Taracila ◽  
Rebecca A. Hutton ◽  
Marion J. Skalweit
Keyword(s):  
Clavulanic Acid ◽  
Susceptibility Testing ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
Oxyanion Hole ◽  
Content Type ◽  
Class A ◽  
Inhibitor Resistance ◽  
Structural Probes ◽  
Variant Residue

ABSTRACTAmbler position 105 in class A β-lactamases is implicated in resistance to clavulanic acid, although no clinical isolates with mutations at this site have been reported. We hypothesized that Y105 is important in resistance to clavulanic acid because changes in positioning of the inhibitor for ring oxygen protonation could occur. In addition, resistance to bicyclic 6-methylidene penems, which are interesting structural probes that inhibit all classes of serine β-lactamases with nanomolar affinity, might emerge with substitutions at position 105, especially with nonaromatic substitutions. All 19 variants of SHV-1 with variations at position 105 were prepared. Antimicrobial susceptibility testing showed thatEscherichia coliDH10B expressing Y105 variants retained activity against ampicillin, except for the Y105L variant, which was susceptible to all β-lactams, similar to the case for the host control strain. Several variants had elevated MICs to ampicillin-clavulanate. However, all the variants remained susceptible to piperacillin in combination with a penem inhibitor (MIC, ≤2/4 mg/liter). The Y105E, -F, -M, and -R variants demonstrated reduced catalytic efficiency toward ampicillin compared to the wild-type (WT) enzyme, which was caused by increasedKm. Clavulanic acid and penemKivalues were also increased for some of the variants, especially Y105E. Mutagenesis at position 105 in SHV yields mutants resistant to clavulanate with reduced catalytic efficiency for ampicillin and nitrocefin, similar to the case for the class A carbapenemase KPC-2. Our modeling analyses suggest that resistance is due to oxyanion hole distortion. Susceptibility to a penem inhibitor is retained although affinity is decreased, especially for the Y105E variant. Residue 105 is important to consider when designing new inhibitors.

scholarly journals Alternative Biotransformation of Retinal to Retinoic Acid or Retinol by an Aldehyde Dehydrogenase from Bacillus cereus

2016 ◽  
Vol 82 (13) ◽  
pp. 3940-3946 ◽  
Author(s):  
Seung-Hye Hong ◽  
Ho-Phuong-Thuy Ngo ◽  
Hyun-Koo Nam ◽  
Kyoung-Rok Kim ◽  
Lin-Woo Kang ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Bacillus Cereus ◽  
Aldehyde Dehydrogenase ◽  
Catalytic Efficiency ◽  
No Oxidation ◽  
Wild Type ◽  
Biotechnological Production ◽  
Oxidation Activity ◽  
Content Type ◽  
Bacterial Enzyme

ABSTRACTA novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified inBacillus cereus. The amino acid sequence of ALDH fromB. cereus(BcALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all-trans-retinal to all-trans-retinoic acid with NAD(P)+. We newly found thatBcALDH and human ALDH (ALDH1A1) could reduce all-trans-retinal to all-trans-retinol with NADPH. The catalytic residues inBcALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency (kcat/Km) for the reduction of all-trans-retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD+converted 400 μM all-trans-retinal to 210 μM all-trans-retinoic acid at the same amount for 240 min, while with NADPH, they converted 400 μM all-trans-retinal to 20, 90, and 40 μM all-trans-retinol, respectively. These results indicate thatBcALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore,BcALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol.IMPORTANCEAlthough mammalian ALDHs have catalyzed the conversion of retinal to retinoic acid with NAD(P)+as a cofactor, a bacterial ALDH involved in the conversion is first characterized. The biotransformation of all-trans-retinal to all-trans-retinoic acid byBcALDH and human ALDH was altered to the biotransformation to all-trans-retinol by a cofactor switch using NADPH. Moreover, the production of all-trans-retinal to all-trans-retinol was changed by mutations at positions 194 and 457 inBcALDH. The alternative biotransformation of retinoids was first performed in the present study. These results will contribute to the biotechnological production of retinoids, including retinoic acid and retinol.

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

scholarly journals Sociality in Escherichia coli: Enterochelin Is a Private Good at Low Cell Density and Can Be Shared at High Cell Density

2015 ◽  
Vol 197 (13) ◽  
pp. 2122-2128 ◽  
Author(s):  
Rebecca L. Scholz ◽  
E. Peter Greenberg
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Iron Acquisition ◽  
Partial Privatization ◽  
High Cell ◽  
Wild Type ◽  
Content Type ◽  
Cell Densities ◽  
The Cost ◽  
Low Cell Density

ABSTRACTMany bacteria produce secreted iron chelators called siderophores, which can be shared among cells with specific siderophore uptake systems regardless of whether the cell produces siderophores. Sharing secreted products allows freeloading, where individuals use resources without bearing the cost of production. Here we show that theEscherichia colisiderophore enterochelin is not evenly shared between producers and nonproducers. Wild-typeEscherichia coligrows well in low-iron minimal medium, and an isogenic enterochelin synthesis mutant (ΔentF) grows very poorly. The enterochelin mutant grows well in low-iron medium supplemented with enterochelin. At high cell densities the ΔentFmutant can compete equally with the wild type in low-iron medium. At low cell densities the ΔentFmutant cannot compete. Furthermore, the growth rate of the wild type is unaffected by cell density. The wild type grows well in low-iron medium even at very low starting densities. Our experiments support a model where at least some enterochelin remains associated with the cells that produce it, and the cell-associated enterochelin enables iron acquisition even at very low cell density. Enterochelin that is not retained by producing cells at low density is lost to dilution. At high cell densities, cell-free enterochelin can accumulate and be shared by all cells in the group. Partial privatization is a solution to the problem of iron acquisition in low-iron, low-cell-density habitats. Cell-free enterochelin allows for iron scavenging at a distance at higher population densities. Our findings shed light on the conditions under which freeloaders might benefit from enterochelin uptake systems.IMPORTANCESociality in microbes has become a topic of great interest. One facet of sociality is the sharing of secreted products, such as the iron-scavenging siderophores. We present evidence that theEscherichia colisiderophore enterochelin is relatively inexpensive to produce and is partially privatized such that it can be efficiently shared only at high producer cell densities. At low cell densities, cell-free enterochelin is scarce and only enterochelin producers are able to grow in low-iron medium. Because freely shared products can be exploited by freeloaders, this partial privatization may help explain how enterochelin production is stabilized inE. coliand may provide insight into when enterochelin is available for freeloaders.

scholarly journals Rational Engineering of Hydratase from Lactobacillus Acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

2019 ◽  
Author(s):  
Bekir Engin Eser ◽  
Michal Poborsky ◽  
Rongrong Dai ◽  
Shigenobu Kishino ◽  
Anita Ljubic ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Substrate Specificity ◽  
Lactobacillus Acidophilus ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Wild Type ◽  
Active Site Residues ◽  
Diverse Range ◽  
Preparative Scale ◽  
Conversion Rates

<div>Enzymatic conversion of abundant fatty acids (FAs) through fatty acid hydratases (FAHs) presents an environment-friendly and efficient route for production of high-value hydroxy fatty acids (HFAs). However, a limited diversity was achieved among HFAs to date with respect to chain length and hydroxy group position, due to high substrate- and regio-selectivity of hydratases. In this study, we compared two highly similar FAHs from <i>Lactobacillus acidophilus</i>: FA-HY2 has narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilize longer chain substrates and hydrate various double bond positions. We reveal three active-site residues that play remarkable role in directing substrate specificity and regioselectivity of hydration. When these residues on FA-HY2 are mutated to the corresponding residues in FA-HY1, we observe a significant expansion of substrate scope and distinct shift and enhancement in hydration of double bonds towards omega-end of FAs. A three-residue mutant of FA-HY2 (TM-FA-HY2; T391S/H393S/I378P) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting ratio of the HFA product regioisomers (10-OH:13-OH) from 99:1 to 12:88. Although kcat values are still low in comparison to wild-type FA-HY1, TM-FA-HY2 exhibited about 60-fold increase in catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) compared to wild-type FA-HY2. Important changes in regioselectivity were also observed with mutant enzymes for arachidonic acid and C18 PUFAs. In addition, TM-FA-HY2 variant exhibited high conversion rates for <i>cis</i>-5, <i>cis</i>-8, <i>cis</i>-11,<i> cis</i>-14, <i>cis</i>-17-eicosapentaenoic acid (EPA) and <i>cis</i>-8, <i>cis</i>-11, <i>cis</i>-14-eicosatrienoic acid (ETA) at preparative scale and enabled isolation of 12-hydroxy products with moderate yields. Furthermore, we demonstrated the potential of microalgae as a source of diverse FAs for HFA production. Our study paves the way for tailor-made FAH design and for efficient conversion of FA sources into diverse range of HFAs with high potential for various applications from polymer industry to medical field.</div><div><br></div>

scholarly journals Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

2019 ◽  
Vol 85 (17) ◽  
Author(s):  
Zhu Jiang ◽  
Tengfei Niu ◽  
Xueqin Lv ◽  
Yanfeng Liu ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Catalytic Efficiency ◽  
Molecular Engineering ◽  
Fine Tuning ◽  
Wild Type ◽  
Content Type ◽  
Chitosan Oligosaccharides ◽  
In The Wild ◽  
Secretory Production ◽  
Extracellular Activity

ABSTRACT Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5′ untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax increased from 5.11 μM s−1 in the wild type to 7.56 μM s−1 in the mutant R157T. IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.

scholarly journals Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

2012 ◽  
Vol 78 (7) ◽  
pp. 2230-2240 ◽  
Author(s):  
Xiaoyun Su ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Catalytic Efficiency ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Ph Optimum ◽  
Content Type ◽  
Caldicellulosiruptor Bescii ◽  
Type Protein ◽  
Wild Type Protein ◽  
Carbohydrate Binding Modules

ABSTRACTThermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses ofCaldicellulosiruptor besciiCel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins ofC. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s−1ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparentkcatof the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in theKmled to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

scholarly journals TetR Family Transcriptional Regulator PccD Negatively Controls Propionyl Coenzyme A Assimilation in Saccharopolyspora erythraea

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Zhen Xu ◽  
Miaomiao Wang ◽  
Bang-Ce Ye
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Coenzyme A ◽  
Transcriptional Regulator ◽  
Fold Increase ◽  
Saccharopolyspora Erythraea ◽  
Wild Type ◽  
Content Type ◽  
Propionate Metabolism ◽  
Key Enzyme

ABSTRACT Propanol stimulates erythromycin biosynthesis by increasing the supply of propionyl coenzyme A (propionyl-CoA), a starter unit of erythromycin production in Saccharopolyspora erythraea. Propionyl-CoA is assimilated via propionyl-CoA carboxylase to methylmalonyl-CoA, an extender unit of erythromycin. We found that the addition of n-propanol or propionate caused a 4- to 16-fold increase in the transcriptional levels of the SACE_3398–3400 locus encoding propionyl-CoA carboxylase, a key enzyme in propionate metabolism. The regulator PccD was proved to be directly involved in the transcription regulation of the SACE_3398–3400 locus by EMSA and DNase I footprint analysis. The transcriptional levels of SACE_3398–3400 were upregulated 15- to 37-fold in the pccD gene deletion strain (ΔpccD) and downregulated 3-fold in the pccD overexpression strain (WT/pIB-pccD), indicating that PccD was a negative transcriptional regulator of SACE_3398–3400. The ΔpccD strain has a higher growth rate than that of the wild-type strain (WT) on Evans medium with propionate as the sole carbon source, whereas the growth of the WT/pIB-pccD strain was repressed. As a possible metabolite of propionate metabolism, methylmalonic acid was identified as an effector molecule of PccD and repressed its regulatory activity. A higher level of erythromycin in the ΔpccD strain was observed compared with that in the wild-type strain. Our study reveals a regulatory mechanism in propionate metabolism and suggests new possibilities for designing metabolic engineering to increase erythromycin yield. IMPORTANCE Our work has identified the novel regulator PccD that controls the expression of the gene for propionyl-CoA carboxylase, a key enzyme in propionyl-CoA assimilation in S. erythraea. PccD represses the generation of methylmalonyl-CoA through carboxylation of propionyl-CoA and reveals an effect on biosynthesis of erythromycin. This finding provides novel insight into propionyl-CoA assimilation, and extends our understanding of the regulatory mechanisms underlying the biosynthesis of erythromycin.

scholarly journals Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

2013 ◽  
Vol 79 (13) ◽  
pp. 4072-4077 ◽  
Author(s):  
Xuguo Duan ◽  
Jian Chen ◽  
Jing Wu
Keyword(s):  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ph Optimum ◽  
Widespread Application ◽  
Content Type ◽  
Major Factors

ABSTRACTPullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase fromBacillus deramificanswere selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that theKmvalues for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and theKcat/Kmvalues increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.

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