Enzymatic treatment to improve permeability and quality of cherry tomatoes for production of dried products.

BACKGROUND: Cherry tomatoes are nutritious and favored by consumers. Processing them into dried cherry tomatoes can prolong their storage life and improve their flavor. The pretreatment of tomato pericarp is crucial for the subsequent processing. However, the traditional physical and chemical treatments of tomato pericarp generally cause nutrient loss and environmental pollution. RESULTS: In this study, a novel enzymatic method for cherry tomatoes was performed using mixed enzymes containing cutinase, cellulase and pectinase. Results showed that the pericarp permeability of cherry tomatoes was effectively improved due to enzymatic treatment. Changes in the microscopic structure and composition of the cuticle were revealed. After treatment with different concentrations of enzymes, cherry tomatoes exhibited higher pericarp permeability and sensory quality to varying degrees. The lycopene content and total polyphenol content significantly increased 2.4- and 1.45-fold, respectively. In addition, the satisfactory effect of the six-time reuse of enzymes on cherry tomatoes could still reach the same level as the initial effect, which effectively reduced the cost of production. CONCLUSIONS: This study revealed for the first time that a mixed enzymatic treatment consisting of cutinase, pectinase and cellulase could effectively degrade the cuticle, enhance the pericarp permeability and improve the quality of cherry tomatoes, with the advantages of being mildly controllable and environmentally friendly, providing a new strategy for the processing of dried cherry tomatoes. © 2023 Society of Chemical Industry.

High-Level Production of Lacto-N-neotetraose in Escherichia coli by Stepwise Optimization of the Biosynthetic Pathway.

Lacto-N-neotetraose (LNnT), an abundant human milk oligosaccharide (HMO), has been approved as a novel functional additive for infant formulas. Therefore, LNnT biosynthesis has attracted extensive attention. Here, a high LNnT-producing, low lacto-N-triose II (LNT II)-residue Escherichia coli strain was constructed. First, an initial LNnT-producing chassis strain was constructed by blocking lactose, UDP-N-acetylglucosamine, and UDP-galactose competitive consumption pathways and introducing ß-1,3-N-acetylglucosaminyltransferase LgtA and ß-1,4-galactosyltransferase LgtB. Subsequently, the supply of LNnT precursors was increased by enhancing UDP-N-acetylglucosamine and UDP-galactose synthesis, inactivating LNT II extracellular transporter SetA, and improving UTP synthesis. Then, modular engineering strategy was used to optimize LNnT biosynthetic pathway fluxes. Moreover, pathway fluxes were fine-tuned by modulating translation initiation strength of essential genes lgtB, prs, and lacY. Finally, LNnT production reached 6.70 g/L in a shake flask and 19.40 g/L in a 3 L bioreactor with 0.47 g/(L h) productivity, with 1.79 g/L LNT II residue, highest productivity level, and lowest LNT II residue thus far.

High-Yield Synthesis of 2'-Fucosyllactose from Glycerol and Glucose in Engineered Escherichia coli.

2'-Fucosyllactose (2'-FL) is vital for the growth and development of newborns. In this study, we developed a synthesis pathway for 2'-FL in Escherichia coli BL21 (DE3). Then, we optimized the solubility of α-1,2-fucosyltransferase, thereby enhancing the production yield of 2'-FL. Based on this finding, we further enhanced the expression of guanosine inosine kinase Gsk and knocked out the isocitrate lyase regulator gene iclR. This strategy reduced the formation of byproduct acetate during the metabolic process and alleviated carbon source overflow effects in the strain, resulting in further improvement of the yield of 2'-FL. In a 3 L bioreactor, employing fed-batch fermentation with glycerol and glucose as substrates, the engineered strain BWLAI-RSZL exhibited impressive 2'-FL titers of 121.9 and 111.56 g/L, along with productivity levels of 1.57 and 1.31 g/L/h, respectively. The reported 2'-FL titers reached a groundbreaking level, irrespective of the carbon source employed (glycerol or glucose), highlighting the significant potential for large-scale industrial synthesis of 2'-FL.

Conformational Switch of the 250s Loop Enables the Efficient Transglycosylation in GH Family 77.

Amylomaltases (AMs) play important roles in glycogen and maltose metabolism. However, the molecular mechanism is elusive. Here, we investigated the conformational dynamics of the 250s loop and catalytic mechanism of Thermus aquaticus TaAM using path-metadynamics and QM/MM MD simulations. The results demonstrate that the transition of the 250s loop from an open to closed conformation promotes polysaccharide sliding, leading to the ideal positioning of the acid/base. Furthermore, the conformational dynamics can also modulate the selectivity of hydrolysis and transglycosylation. The closed conformation of the 250s loop enables the tight packing of the active site for transglycosylation, reducing the energy penalty and efficiently preventing the penetration of water into the active site. Conversely, the partially closed conformation for hydrolysis results in a loosely packed active site, destabilizing the transition state. These computational findings guide mutation experiments and enable the identification of mutants with an improved disproportionation/hydrolysis ratio. The present mechanism is in line with experimental data, highlighting the critical role of conformational dynamics in regulating the catalytic reactivity of GHs.

Differentiated digestion resistance and physicochemical properties of linear and α-1,2/α-1,3 branched isomaltodextrins prepared by 4,6-α-glucanotransferase and branching sucrases.

Isomaltodextrins (IMDs) are starch-based dietary fibers (DF) prepared enzymatically, which show great potential as a functional food ingredient. In this study, a series of novel IMDs with diverse structures were generated by 4,6-α-glucanotransferase GtfBΔN from Limosilactobacillus fermentum NCC 3057, combined with two α-1,2 and α-1,3 branching sucrases. Results indicated that α-1,2 and α-1,3 branching significantly improved the DF contents of α-1,6 linear products up to 60.9-62.8%. When altering the ratios of [sucrose]/[maltodextrin], IMDs containing 25.8-89.0% α-1,6 bonds, 0-59.6% α-1,2 bonds and 0-35.1% α-1,3 bonds and Mw ranged from 1967 to 4876 Da were obtained. Physicochemical property analysis showed that grafting with α-1,2 or α-1,3 single glycosyl branches can improve the solubility of the α-1,6 linear product, in which α-1,3 branched products were better. Moreover, α-1,2 or α-1,3 branching did no effect on the viscosity of the products but Mw did, the larger Mw the greater viscosity. In addition, α-1,6 linear and α-1,2 or α-1,3 branched IMDs all exhibited strong acid-heating stabilities, freeze-thaw stabilities, and good resistance to browning caused by the Maillard reaction. Branched IMDs showed excellent storage stabilities at room temperature for one year at a concentration of 60%, whereas 45% α-1,6 linear IMD precipitated quickly within 12 h. Most importantly, α-1,2 or α-1,3 branching remarkably increased the contents of resistant starch in the α-1,6 linear IMDs to 74.5-76.8%. These clear qualitative assessments demonstrated the outstanding processing and application properties of the branched IMDs and were expected to provide valuable perspectives toward the technological innovation of functional carbohydrates.

Structural characterization of slow digestion dextrin synthesized by a combination of α-glucosidase and cyclodextrin glucosyltransferase and its prebiotic potential on the gut microbiota in vitro.

Starch-based dietary fibers are at the forefront of functional ingredient research. In this study, a novel water-soluble slow digestion dextrin (SDD) was synthesized by synergy of α-glucosidase and cyclodextrin glucosyltransferase and characterized. Results showed that SDD exhibited high solubility, low viscosity, and resistance to digestive enzymes, and also showed an increased dietary fiber content of 45.7% compared with that of α-glucosidase catalysis alone. Furthermore, SDD was used as the sole carbon source to ferment selected intestinal strains and human fecal microflora in vitro to investigate its prebiotic effects. It was found that SDD could markedly enriched the abundance of Bifidobacterium, Veillonella, Dialister, and Blautia in human gut microflora and yielded higher total organic acid. The combination of α-glucosidase and cyclodextrin glucosyltransferase in this study showed valuable potential for the preparation of a novel slow digestion dextrin with good physicochemical properties and improved prebiotic effects.

Mechanistic Insights into How the Protonation State of D234 Dictates the Reactivity in Streptomyces coelicolor ß-N-Acetylhexosaminidase.

ß-N-Acetylhexosaminidases (HEXs) play important roles in human diseases and the biosynthesis of human milk oligosaccharides. Despite extensive research, the catalytic mechanism of these enzymes remains largely unexplored. In this study, we employed quantum mechanics/molecular mechanics metadynamics to investigate the molecular mechanism of Streptomyces coelicolor HEX (ScHEX), which has shed light on the transition state structures and conformational pathways of this enzyme. Our simulations revealed that Asp242, located near the assisting residue, can switch the reaction intermediate to an oxazolinium ion or a neutral oxazoline, depending on the protonation state of the residue. Moreover, our findings indicated that the free energy barrier of the second-step reaction starting from the neutral oxazoline increases steeply due to the reduction in the anomeric carbon positive charge and the shortening of the C1-O2N bond. Our results provide valuable insights into the mechanism of substrate-assisted catalysis and could facilitate the design of inhibitors and the engineering of analogous glycosidases for biosynthesis.

Enhancing 2-O-α-D-glucopyranosyl-L-ascorbic acid synthesis by weakening the acceptor specificity of CGTase toward glucose and maltose.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid (L-AA), which has been widely used in food and cosmetics industries. Sugar molecules, such as glucose and maltose produced by cyclodextrin glycosyltransferase (CGTase) during AA-2G synthesis may compete with L-AA as the acceptors, resulting in low AA-2G yield. Multiple sequence alignment combined with structural simulation analysis indicated that residues at positions 191 and 255 of CGTase may be responsible for the difference in substrate specificity. To investigate the effect of these two residues on the acceptor preference and the AA-2G yield, five single mutants Bs F191Y, Bs F255Y, Bc Y195F, Pm Y195F and Pm Y260F of three CGTases from Bacillus stearothermophilus NO2 (Bs), Bacillus circulans 251 (Bc) and Paenibacillus macerans (Pm) were designed for AA-2G synthesis. Under optimal conditions, the AA-2G yields of the mutants Bs F191Y and Bs F255Y AA-2G were 34.3% and 7.9% lower than that of Bs CGTase, respectively. The AA-2G yields of mutant Bc Y195F, Pm Y195F and Pm Y260F were 45.8%, 36.9% and 12.6% higher than those of wild-type CGTases, respectively. Kinetic studies revealed that the residues at positions 191 and 255 of the three CGTases were F, which decreased glucose and maltose specificity and increased L-AA specificity. This study not only proposes for the first time that the AA-2G yield can be improved by weakening the acceptor specificity of CGTase toward sugar byproducts, but also provides new insight on the modification of CGTase that catalyze the double-substrate transglycosylation reaction.

Multiple approaches of loop region modification for thermostability improvement of 4,6-α-glucanotransferase from Limosilactobacillus fermentum NCC 3057.

4,6-α-glucanotransferase (4,6-α-GT), as a member of the glycoside hydrolase 70 (GH70) family, converts starch/maltooligosaccharides into α,1-6 bond-containing α-glucan and possesses potential applications in food, medical and related industries but does not satisfy the high-temperature requirement due to its poor thermostability. In this study, a 4,6-α-GT (ΔGtfB) from Limosilactobacillus fermentum NCC 3057 was used as a model enzyme to improve its thermostability. The loops of ΔGtfB as the target region were optimized using directed evolution, sequence alignment, and computer-aided design. A total of 11 positive mutants were obtained and iteratively combined to obtain a combined mutant CM9, with high resistance to temperature (50 °C). The activity of mutant CM9 was 2.08-fold the activity of the wild type, accompanied by a 5 °C higher optimal temperature, a 5.76 °C higher melting point (Tm, 59.46 °C), and an 11.95-fold longer half-life time (t1/2). The results showed that most of the polar residues in the loop region of ΔGtfB are mutated into rigid proline residues. Molecular dynamics simulation demonstrated that the root mean square fluctuation of CM9 significantly decreased by "Breathing" movement reduction of the loop region. This study provides a new strategy for improving the thermostability of 4,6-α-GT through rational loop region modification.

Producing 2-O-α-D-glucopyranosyl-L-ascorbic acid by modified cyclodextrin glucosyltransferase and isoamylase.

In this study, site saturation mutagenesis was performed on the - 3 (R44, D86, S90, and D192) and - 6 subsite (Y163, G175, G176, and N189) of Bacillus stearothermophilus NO2 cyclodextrin glucosyltransferase to enhance its specificity for the donor substrate maltodextrin for 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) preparation. The AA-2G yields produced by the mutants S90D, G176H, and S90D/G176H were 181, 171, and 185 g/L, respectively. Our previous study found that the mutant K228R/M230L also increased the AA-2G yield. Therefore, the mutants S90D, G176H, S90D/G176H, and K228R/M230L were further used to generate combinatorial mutants. Among these mutants, the highest AA-2G yield (217 g/L) was produced by S90D/K228R/M230L with 500 g/L maltodextrin as the glucosyl donor, which was 56 g/L higher than that produced by wild-type CGTase. In addition, AA-2G was prepared by adding isoamylase to hydrolyze α-1,6 glucosidic linkages in maltodextrin that could not be utilized by CGTase to improve the utilization rate of maltodextrin. The addition of isoamylase reduced the concentration of maltodextrin from 500 to 350 g/L, while the AA-2G yield remained high (208 g/L). The preparation of AA-2G by complexing isoamylase with mutant S90D/K228R/M230L reduced the maltodextrin concentration by 150 g/L, while the AA-2G yield increased by 47 g/L than preparation with wild-type CGTase alone, which laid a foundation for the large-scale preparation of AA-2G. KEY POINTS: • Mutants exhibited improved maltodextrin specificity. • Mutant S90D/K228R/M230L produced high yield of AA-2G with maltodextrin as substrate. • AA-2G was first synthesized by a combination of isoamylase and CGTase.

Oxidative degradation of pre-oxidated polystyrene plastics by dye decolorizing peroxidases from Thermomonospora curvata and Nostocaceae.

Biodegradation of PS has attracted lots of public attentions due to its environmental friendliness. However, no specific PS degrading enzyme has been identified yet. Dye decolorizing peroxidases (DyPs) are heme-containing peroxidases named for the ability to degrade a variety of organic dyes. Herein, the abilities of two DyPs from Thermomonospora curvata (TcDyP) and Nostocaceae (AnaPX) to degrade PS were evaluated. Preoxidation methods by ultraviolet (UV) irradiation and chemical oxidants were developed to initially activate C-C bonds in the PS skeleton. DyPs degradation caused obvious etching and enhanced hydrophilicity of UV-PS films, and also generated new CO and C-OH groups. The cleavage of activated C-C bonds by DyPs was experimentally proven by analyzing the degradation products of UV-PS and model substrates. Furthermore, better pre-oxidation was obtained by using chemical oxidants KMnO4/H2SO4 and mCPBA to oxidize PS materials in dissolved state. And AnaPX exhibited stronger degradation effects on KMnO4/H2SO4-PS and mCPBA-PS by causing greater changes in functional groups CO, C-O, -OH groups and substituted benzenes and higher molecular weight reductions of 19.7% and 31.0%, respectively. To our knowledge, this is the first report on the identification of PS-degrading enzymes that provides experimental evidence.

A Single Hydrogen Bond Controls the Selectivity of Transglycosylation vs Hydrolysis in Family 13 Glycoside Hydrolases.

Converting glycoside hydrolases (GHs) from hydrolytic to synthetic enzymes via transglycosylation is a long-standing goal for the biosynthesis of complex carbohydrates. However, the molecular determinants for the selectivity of transglycosylation (T) vs hydrolysis (H) are still not fully unraveled. Herein, we show experimentally that mutation of one active site residue can switch the enzyme activity between hydrolysis and transglycosylation in two highly homologous GHs. Further QM/MM simulations reveal that the mutation modulates the T vs H reaction barriers via the presence/absence of a single H-bond with the nucleophile Asp. Such a H-bond controls the product selectivity via a dual effect: on one hand, it facilitates the breaking of the glycosyl-enzyme intermediate. On the other, it displaces the sugar acceptor, resulting in a reduced affinity and significant steric repulsion for transglycosylation. These findings expand our understanding of the molecular mechanisms that modulate the T/H balance in GHs.

Trehalose promotes high-level heterologous expression of 4,6-α-glucanotransferase GtfR2 in Escherichia coli and mechanistic analysis.

4,6-α-Glucanotransferases (4,6-α-GTs) hold great potential for applications in the food and medical industries because of their efficient transglycosylation ability. However, it is relatively difficult to achieve high soluble expression because of their high molecular weight and multidomain nature. In this study, 4,6-α-GT of Burkholderia sp. (GtfR2) was successfully expressed in E. coli, and the activity attained 1.55 × 104 U/mL by traditional fermentation optimization. However, a large number of inactive inclusion bodies of GtfR2 were still present due to aggregation and precipitation. The trehalose-mediated strategy was first proposed and applied in the fermentation process of GtfR2. Trehalose addition significantly reduced inclusion bodies, resulting in an increase in GtfR2 activity (6.48 × 104 U/mL), which was 4.20 times higher than that of the control group. Our molecular dynamics simulations revealed that trehalose could spontaneously stabilize the conformational dynamics of GtfR2 by binding to the groove, loop, α-helix and N-terminal unstable regions on the surface. This strategy was also available to enhance the soluble expression of other 4,6/4,3-α-GTs, which were increased by 3.03-77.19 times. This study is the first to observe that trehalose can inhibit the aggregation and precipitation of GtfR2, which provides a new perspective for the recombinant expression of 4,6/4,3-α-GTs.

Enhanced extracellular α-amylase production in Brevibacillus choshinensis by optimizing extracellular degradation and folding environment.

A strategy for optimizing the extracellular degradation and folding environment of Brevibacillus choshinensis has been used to enhance the extracellular production of recombinant α-amylase. First, a gene (bcp) encoding an extracellular protease and another encoding an extracellular chaperone (prsC) were identified in the genome of B. choshinensis HPD31-SP3. Then, the effect of extracellular protein degradation on recombinant α-amylase production was investigated by establishing a CRISPR/Cas9n system to knock out bcp. The effect of extracellular folding capacity was investigated separately by coexpressing extracellular chaperones genes from different sources (prsA, prsC, prsL, prsQ) in B. choshinensis. The final recombinant strain (BCPPSQ), which coexpressed prsQ in a genetic background lacking bcp, produced an extracellular α-amylase activity of 6940.9 U/ml during shake-flask cultivation. This was 2.1-fold greater than that of the original strain BCWPS (3367.9 U/ml). Cultivation of BCPPSQ in a 3-l fermenter produced an extracellular α-amylase activity of 17925.6 U/ml at 72 h, which was 7.6-fold greater than that of BCWPS (2358.1 U/ml). This strategy demonstrates its great potential in enhancing extracellular α-amylase production in B. choshinensis. What's more, this study provides a strategic reference for improving the extracellular production of other recombinant proteins in B. choshinensis.

Enhancing Extracellular Pullulanase Production in Bacillus subtilis Through dltB Disruption and Signal Peptide Optimization.

Bacillus subtilis has many attributes that make it a popular host for recombinant protein production. Although its protein production ability has been enhanced through protease gene disruption, residual proteases like quality control HtrA and HtrB can limit protein yield by degrading inadequately folded proteins present during overexpression. In this study, two strategies were employed to increase production of industrial enzyme pullulanase: enhancing extracellular pullulanase folding and optimizing its signal peptide. The hypothesis was that disruption of dltB gene of expression host B. subtilis WS9 would enhance recombinant extracellular folding by increasing cation binding to the cell's outer envelope. Consistent with this hypothesis, disrupting dltB enhanced pullulanase production by 49% in shake-flask cultures. The disruption also enhanced extracellular α-CGTase and ß-CGTase production by 25% and 35%, respectively. Then, more effective signal peptide for pullulanase production was identified through high-throughput screening of 173 unique B. subtilis signal peptides. Replacing the native signal peptide of pullulanase with that encoded by ywtF increased extracellular pullulanase activity by an additional 12%. Three-liter fermenter scale-up production yielded the highest extracellular pullulanase activity reported to date: 8037.91 U·mL-1. This study highlights the usefulness of dltB deletion and signal peptide optimization in enhancing extracellular protein production.

Directed Mutation of Two Key Amino Acid Residues Alters the Product Structure of the New 4,6-α-Glucanotransferase from Bacillus sporothermodurans.

4,6-α-Glucanotransferases (4,6-α-GTs) convert amylose V into two types of differently structured products: a linear product connected by continuous α,1 → 6 bonds, such as isomalto/malto-polysaccharide (IMMP), and a highly branched product connected by alternating α,1 → 4 and α,1 → 6 bonds, such as reuteran-like polysaccharide (RLP). The synthesis process of 4,6-α-GT products is unclear, and exploring this process is significant for producing dietary fibers with potential applications. This study identified and expressed Geobacillus sp. 12AMOR1 GtfD-ΔC and Bacillus sporothermodurans GtfC-ΔC. After characterizing their products through 1H NMR and enzymatic fingerprinting, we found that GtfD-ΔC synthesized RLP with 29% α,1 → 6 bonds, and GtfC-ΔC synthesized IMMP with 71% α,1 → 6 bonds. The maltoheptaose incubation experiment showed different chain-length transfer patterns of two 4,6-α-GTs, GtfC-ΔC and GtfD-ΔC, transferring single and multiple glucose residues in each transglycosylation reaction, respectively. Site-directed mutagenesis confirmed that positions S345 and I347 influence the product structure of GtfC-ΔC, and the S345T/I347V mutation changed the GtfC-ΔC product to a linear product connected by alternating α,1 → 4 and α,1 → 6 bonds (pullulan-like polysaccharide) and altered the chain-length transfer pattern of GtfC-ΔC. We proposed that different chain-length transfer patterns between GtfD-ΔC and GtfC-ΔC may explain their differences in product structures. These findings are significant for obtaining the desired dietary fiber by engineering 4,6-α-GT.

Microbial starch debranching enzymes: Developments and applications.

Starch debranching enzymes (SDBEs) hydrolyze the α-1,6 glycosidic bonds in polysaccharides such as starch, amylopectin, pullulan and glycogen. SDBEs are also important enzymes for the preparation of sugar syrup, resistant starch and cyclodextrin. As the synergistic catalysis of SDBEs and other starch-acting hydrolases can effectively improve the raw material utilization and production efficiency during starch processing steps such as saccharification and modification, they have attracted substantial research interest in the past decades. The substrate specificities of the two major members of SDBEs, pullulanases and isoamylases, are quite different. Pullulanases generally require at least two α-1,4 linked glucose units existing on both sugar chains linked by the α-1,6 bond, while isoamylases require at least three units of α-1,4 linked glucose. SDBEs mainly belong to glycoside hydrolase (GH) family 13 and 57. Except for GH57 type II pullulanse, GH13 pullulanases and isoamylases share plenty of similarities in sequence and structure of the core catalytic domains. However, the N-terminal domains, which might be one of the determinants contributing to the substrate binding of SDBEs, are distinct in different enzymes. In order to overcome the current defects of SDBEs in catalytic efficiency, thermostability and expression level, great efforts have been made to develop effective enzyme engineering and fermentation strategies. Herein, the diverse biochemical properties and distinct features in the sequence and structure of pullulanase and isoamylase from different sources are summarized. Up-to-date developments in the enzyme engineering, heterologous production and industrial applications of SDBEs is also reviewed. Finally, research perspective which could help understanding and broadening the applications of SDBEs are provided.

Effect of Leu277 on Disproportionation and Hydrolysis Activity in Bacillus stearothermophilus NO2 Cyclodextrin Glucosyltransferase.

The disproportionation activity of cyclodextrin glucosyltransferase (CGTase; EC 2.4.1.19) can be used to convert small molecules into glycosides, thereby enhancing their solubility and stability. However, CGTases also exhibit a competing hydrolysis activity. The +2 subsite of the substrate binding cleft plays an important role in both the disproportionation and hydrolysis activities, but almost all known mutations at this site decrease disproportionation activity. In this study, Leu277 of the CGTase from Bacillus stearothermophilus NO2, located near both the +2 subsite and the catalytic acid/base Glu253, was modified to assess the effect of side chain size at this position on disproportionation and hydrolysis activities. The best mutant, L277M, exhibited a reduced Km for the acceptor substrate maltose (0.48 mM versus 0.945 mM) and an increased kcat/Km (1,175 s-1 mM-1 versus 686.1 s-1 mM-1), compared with those of the wild-type enzyme. The disproportionation-to-hydrolysis ratio of L277M was 2.4-fold greater than that of the wild type. Existing structural data were combined with a multiple-sequence alignment and Gly282 mutations to examine the mechanism behind the effects of the Leu277mutations. The Gly282 mutations were included to aid a molecular dynamics (MD) analysis and the comparison of crystal structures. They reveal that changes to a hydrophobic cluster near Glu253 and the hydrophobicity of the +2 subsite combine to produce the observed effects. IMPORTANCE In this study, mutations that enhance the disproportionation to hydrolysis ratio of a CGTase have been discovered. For example, the disproportionation-to-hydrolysis ratio of the L277M mutant of Bacillus stearothermophilus NO2 CGTase was 2.4-fold greater than that of the wild type. The mechanism behind the effects of these mutations is explained. This paper opens up other avenues for future research into the disproportionation and hydrolysis activities of CGTases. Productive mutations are no longer limited to the acceptor subsite, since mutations that indirectly affect the acceptor subsite also enhance enzymatic activity.

Enhanced Production of Soluble Pyrococcus furiosus α-Amylase in Bacillus subtilis through Chaperone Co-Expression, Heat Treatment and Fermentation Optimization.

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100°C), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90°C for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

Efficient secretory expression of Bacillus stearothermophilus α/ß-cyclodextrin glycosyltransferase in Bacillus subtilis.

Bacillus stearothermophilus α/ß-cyclodextrin glycosyltransferase (α/ß-CGTase) is an excellent transglycosylase with broad potential for food application, but its expression level is low in Bacillus subtilis. In this study, the optimal signal peptide for α/ß-CGTase expression was screened from 173 signal peptides in B. subtilis WS11. The α/ß-CGTase activity in a 3-L fermentor reached 151.93 U⋅ mL-1, but substantial amounts of inclusion bodies were produced. The N-terminal 12 amino acids of α/ß-CGTase were then replaced with the N-terminal 15 amino acids of a ß-CGTase from the same family that has a high percentage of disorder-promoting amino acids. As a result, the inclusion bodies were significantly reduced, and the enzyme activity increased to 249.35 U mL-1, 2.3 times that of the strain constructed previously. Finally, the ppsE and sfp genes of B. subtilis WS11, which are related to lipopeptide biosurfactant synthesis, were knocked out to produce B. subtilis WS13. When B. subtilis WS13 was used to produce α/ß-CGTase in a 3-L fermentor, 70 % less defoaming agent was required than with B. subtilis WS11. Furthermore, enzyme production and growth of WS13 were equivalent to those of WS11. This study is of great significance for future research to efficiently scale-up production of α/ß-CGTase.