CRISPRi screen for enhancing heterologous α-amylase yield in Bacillus subtilis.

Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two α-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3' UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.

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.

Optimization and scale-up of α-amylase production by Aspergillus oryzae using solid-state fermentation of edible oil cakes.

BACKGROUND: Amylases produced by fungi during solid-state fermentation are the most widely used commercial enzymes to meet the ever-increasing demands of the global enzyme market. The use of low-cost substrates to curtail the production cost and reuse solid wastes are seen as viable options for the commercial production of many enzymes. Applications of α-amylases in food, feed, and industrial sectors have increased over the years. Additionally, the demand for processed and ready-to-eat food has increased because of the rapid growth of food-processing industries in developing economies. These factors significantly contribute to the global enzyme market. It is estimated that by the end of 2024, the global α-amylase market would reach USD 320.1 million (Grand View Research Inc., 2016). We produced α-amylase using Aspergillus oryzae and low-cost substrates obtained from edible oil cake, such as groundnut oil cake (GOC), coconut oil cake (COC), sesame oil cake (SOC) by solid-state fermentation. We cultivated the fungus using these nutrient-rich substrates to produce the enzyme. The enzyme was extracted, partially purified, and tested for pH and temperature stability. The effect of pH, incubation period and temperature on α-amylase production using A. oryzae was optimized. Box-Behnken design (BBD) of response surface methodology (RSM) was used to optimize and determine the effects of all process parameters on α-amylase production. The overall cost economics of α-amylase production using a pilot-scale fermenter was also studied. RESULTS: The substrate optimization for α-amylase production by the Box-Behnken design of RSM showed GOC as the most suitable substrate for A. oryzae, as evident from its maximum α-amylase production of 9868.12 U/gds. Further optimization of process parameters showed that the initial moisture content of 64%, pH of 4.5, incubation period of 108 h, and temperature of 32.5 °C are optimum conditions for α-amylase production. The production increased by 11.4% (10,994.74 U/gds) by up-scaling and using optimized conditions in a pilot-scale fermenter. The partially purified α-amylase exhibited maximum stability at a pH of 6.0 and a temperature of 55 °C. The overall cost economic studies showed that the partially purified α-amylase could be produced at the rate of Rs. 622/L. CONCLUSIONS: The process parameters for enhanced α-amylase secretion were analyzed using 3D contour plots by RSM, which showed that contour lines were more oriented toward incubation temperature and pH, having a significant effect (p < 0.05) on the α-amylase activity. The optimized parameters were subsequently employed in a 600 L-pilot-scale fermenter for the α-amylase production. The substrates were rich in nutrients, and supplementation of nutrients was not required. Thus, we have suggested an economically viable process of α-amylase production using a pilot-scale fermenter.

The Yeast eIF2 Kinase Gcn2 Facilitates H2O2-Mediated Feedback Inhibition of Both Protein Synthesis and Endoplasmic Reticulum Oxidative Folding during Recombinant Protein Production.

Recombinant protein production is a known source of oxidative stress. However, knowledge of which reactive oxygen species are involved or the specific growth phase in which stress occurs remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, microcultivation, and continuous measurements in batch culture, we observed H2O2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H2O2, we find that Gcn2-dependent phosphorylation of eIF2α increases alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains but not in the wild type. Gcn2 deficiency furthermore reduced intracellular H2O2 levels and the Hac1 splicing ratio, while expression of antioxidants and the endoplasmic reticulum (ER) disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding are coupled and subject to feedback inhibition by H2O2. IMPORTANCE Recombinant protein production is a multibillion dollar industry. Optimizing the productivity of host cells is, therefore, of great interest. In several hosts, oxidants are produced as an unwanted side product of recombinant protein production. The buildup of oxidants can result in intracellular stress responses that could compromise the productivity of the host cell. Here, we document a novel protein synthesis inhibitory mechanism that is activated by the buildup of a specific oxidant (H2O2) in the cytosol of yeast cells upon the production of recombinant proteins. At the center of this inhibitory mechanism lies the protein kinase Gcn2. By removing Gcn2, we observed a doubling of recombinant protein productivity in addition to reduced H2O2 levels in the cytosol. In this study, we want to raise awareness of this inhibitory mechanism in eukaryotic cells to further improve protein production and contribute to the development of novel protein-based therapeutic strategies.

How Different Snacks Produce a Distinct Effect in Salivary Protein Composition.

Saliva secretion changes in response to different stimulation. Studies performed in animals and humans suggest that dietary constituents may influence saliva composition, although the dynamics of these changes, and how they are specific for each type of food, are little known. The objective of the present study was to access the short-term effects of different foods in salivation and salivary protein composition. Twelve participants were tested for four snacks (yoghurt, bread, apple and walnuts). Non-stimulated saliva was collected before and at 0', 5' and 30' after each snack intake. Flow rate, total protein, alpha-amylase enzymatic activity and salivary protein profile were analyzed. Yoghurt and apple were the snacks resulting in higher salivary changes, with higher increases in flow rate and alpha-amylase activity immediately after intake. The expression levels of immunoglobulin chains decreased after the intake of all snacks, whereas cystatins and one pink band (proline-rich proteins-PRPs) increased only after yoghurt intake. Walnut's snack was the one resulting in lower changes, probably due to lower amounts eaten. Even so, it resulted in the increase in one PRPs band. In conclusion, changes in saliva composition varies with foods, with variable changes in proteins related to oral food processing and perception.

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.

Biosynthesis and industrial applications of α-amylase: a review.

Amylase is amongst the most indispensable enzymes that have a large number of applications in laboratories and industries. Mostly, α-amylase is synthesized from microbes such as bacteria, fungi and yeast. Due to the high demand for α-amylase, its synthesis can be enhanced using recombinant DNA technology, different fermentation methods, less expensive and good carbon and nitrogen sources, and optimizing the various parameters during fermentation, e.g., temperature, pH and fermentation duration. Various methods are used to measure the production and activity of synthesized α-amylase like iodine, DNS, NS and dextrinizing methods. The activity of crude α-amylase can be elevated to the maximum level by optimizing the temperature and pH. Some metals also interact with α-amylase and increase its activity like K+, Na+, Mg2+ and Ca2+. Some industries such as starch conversion, food, detergent, paper, textile industries and fuel alcohol production extensively utilize α-amylase for their various purposes.

Plasma-mediated enhancement of enzyme secretion in Aspergillus oryzae.

Technical bottlenecks in protein production and secretion often limit the efficient and robust industrial use of microbial enzymes. The potential of non-thermal atmospheric pressure plasma to overcome these technical barriers was examined. Spores of the fermenting fungus Aspergillus oryzae (A. oryzae) were submerged in potato dextrose broth (PDB) (5 × 106 per ml) and treated with micro dielectric barrier discharge plasma at an input voltage of 1.2 kV and current of 50 to 63 mA using nitrogen as the feed gas. The specific activity of α-amylase in the broth was increased by 7.4 to 9.3% after 24 and 48 h of plasma treatment. Long-lived species, such as NO2 - and NO3 - , generated in PDB after plasma treatment may have contributed to the elevated secretion of α-amylase. Observations after 24 h of plasma treatment also included increased accumulation of vesicles at the hyphal tip, hyphal membrane depolarization and higher intracellular Ca2+ levels. These results suggest that long-lived nitrogen species generated in PDB after plasma treatment can enhance the secretion of α-amylase from fungal hyphae by depolarizing the cell membrane and activating Ca2+ influx into hyphal cells, eventually leading to the accumulation of secretory vesicles near the hyphal tips.

Purification and characterization of thermostable α-amylase produced from Bacillus licheniformis So-B3 and its potential in hydrolyzing raw starch.

AIMS: This work was achieved to obtain the optimum culture conditions of the thermostable alpha-amylase produced by thermophilic Bacillus licheniformis SO-B3. Furthermore, the α-amylase was purified and then characterized, and also its kinetic parameters were determined. MATERIALS AND METHODS: A new thermotolerant bacteria called Bacillus licheniformis SO-B3 employed in this work was isolated from a sample of thermal spring mud in Sirnak (Meyremderesi). Several parameters such as the impact of temperature, time, and pH on enzyme production were examined. Thin-Layer Chromatography (TLC) was employed to analyze the end-products of soluble starch hydrolysis, and the utilization of purified α-amylase in the clarification of unripe apple juices was studied. KEY FINDINGS: The highest enzyme production conditions were determined as 35 °C, 36th hour, and pH 7.0. Thermostable α-amylase was purified by 70% ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and dialysis, with a 51-purification fold and 30% yield recovery. The Km and Vmax values for this enzyme were 0.004 mM and 3.07 µmol min-1 at 70 °C, respectively. The α-amylase's molecular weight was found as 74 kDa. In addition, α-amylase showed a good degradation rate for raw starch. SIGNIFICANCE: It was hypothesized that Bacillus licheniformis SO-B3 could be used as an α-amylase source. These findings displayed that purified enzyme could be utilized in fruit juice industries for clarification of apple juice and raw starch hydrolyzing.

Reducing the cell lysis to enhance yield of acid-stable alpha amylase by deletion of multiple peptidoglycan hydrolase-related genes in Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens is a major industrial host for extracellular protein production, with great potential in the enzyme industry. However, the strain has accelerated the autolysis drawback in the process of secreting extracellular enzymes, which can significantly lower the density of cells and decrease the product yield. To identify target genes, we employed comparative transcriptome sequencing and KEGG analysis to indicate the increased expression of peptidoglycan hydrolase-regulated genes from the exponential phase to the apoptotic phase of growth; this was further confirmed by quantitative RT-PCR. By deleting lytD, lytE, and sigD genes, cell lysis was reduced and the production of acid-stable Bacillus licheniformis alpha-amylase was enhanced. After 36 h of culture, multiple deletion mutant BA ΔSDE had significantly more viable cells compared to the control strain BA Δupp, and flow cytometry analysis indicated that 48.43% and 64.03% of the cells were lysed in cultures of BA ΔSDE and BA Δupp, respectively. In a 2-L fed-batch fermenter, viable cell number of the triple deletion mutant BA ΔSDE increased by 2.79 Log/cfu/mL, and the activity of acid-stable alpha-amylase increased by 48.4%, compared to BA Δupp. Systematic multiple peptidoglycan hydrolases deletion relieved the autolysis and increased the production of industrial enzymes, and provided a useful strategy for guiding efforts to manipulate the genomes of other B. amyloliquefaciens used for chassis host.

Multiplex genetic engineering improves endogenous expression of mesophilic α-amylase gene in a wild strain Bacillus amyloliquefaciens 205.

A wild strain Bacillus amyloliquefaciens 205 was screened for its high activity of α-amylase. A mesophilic α-amylase encoding gene amyE-205 was revealed and analyzed by genome sequencing. In order to facilitate plasmid transformation to strain 205, an interspecific plasmid transformation method was improved with 5-13 times higher in transformants than that of electronic transformation. A series of CRISPR genome editing tools have been successfully constructed for gene knockout, transcript repression and activation in 205 genome. At this basis, sporulation related genes spo0A and spoIIAC were knockout and suppressed with CRISPR/Cas9 and CRISPR/dCas9 respectively. The double knockout strain 205spo- was eliminated sporulation with 22.8% increasing of α-amylase activity. The optimal binding site G8 for dCas9-ω has been confirmed in the transcript activation. When amyE-205 was over-expressed with high copy plasmid pUC980-2, its whole upstream sequences containing G8 were also cloned. Whereafter, dCas9-ω was used to activate amyE-205 expression both at genome and plasmid. The final engineered strain 205PG8spo- achieved 784.3% promotion on α-amylase activity than the starting strain 205. The novel genetic tool box containing an efficient interspecific transformation method and functional CRISPR systems, superadded the multiplex regulation strategies used in strain modification would be also applicative in many Bacillus species.

Evaluating and engineering Saccharomyces cerevisiae promoters for increased amylase expression and bioethanol production from raw starch.

Bioethanol production from starchy biomass via consolidated bioprocessing (CBP) will benefit from amylolytic Saccharomyces cerevisiae strains that produce high levels of recombinant amylases. This could be achieved by using strong promoters and modification thereof to improve gene expression under industrial conditions. This study evaluated eight endogenous S. cerevisiae promoters for the expression of a starch-hydrolysing α-amylase gene. A total of six of the native promoters were modified to contain a promoter-proximal intron directly downstream of the full-length promoter. Varying results were obtained; four native promoters outperformed the ENO1P benchmark under aerobic conditions and two promoters showed better expression under simulated CBP conditions. The addition of the RPS25A intron significantly improved the expression from most promoters, displaying increased transcript levels, protein concentrations and amylase activities. Raw starch-utilising strains were constructed through co-expression of selected α-amylase cassettes and a glucoamylase gene. The amylolytic strains displayed improved fermentation vigour on raw corn starch and broken rice, reaching 97% of the theoretical ethanol yield and converting 100% of the available carbon to products within 120 h in small-scale CBP fermentations on broken rice. This study showed that enhanced amylolytic strains for the conversion of raw starch to ethanol can be achieved through turnkey promoter selection and/or engineering.

Production, purification and applications of raw starch degrading and calcium-independent α-amylase from soil rich in extremophile.

Calcium independent, raw starch hydrolyzing, acidic α-amylase (66 kDa) was synthesized by Bacillus subtilis S113 that is an aerobic, rod-shaped and Gram +ve bacteria. Purification of the enzyme was performed by HiTrap Capto Q (Ion-exchange chromatography; 19.28 fold; 22.41% yield). The purified enzyme was found stable at broad acidic pH (4-6.5) and high-temperature range (40-80 °C), that fulfilled the necessary criteria and laid the foundation to be utilized in starch saccharification industry. Kinetic studies of the enzyme revealed that Km and Vmax of the enzyme was 0.22% and 357.14 U/mg respectively. Scanning electron microscopy studies showed that the enzyme was capable of completely hydrolyzing raw wheat and potato starch, further confirming its role in the starch industry. It was found that only 7.93% of the activity was loss at 4 °C when kept for one year.

Production, characterization and application of thermostable, alkaline α-amylase (AA11) from Bacillus cereus strain SP-CH11 isolated from Chilika Lake.

An alkaliphile bacterial strain designated as CH11 was isolated from the sediments of Chilika Lake, Odisha. The isolate showed stupendous growth and production of α-amylase at pH 10.0. Through 16S rRNA gene based molecular technique this isolate was identified as Bacillus cereus strain SP-CH11 having GenBank Accession No. KT992791. Homogenous ~55 kDa extracellular α-amylase was extracted with 241.304, 26.26 and 3.2-fold acceleration in specific activity, purification fold and yield respectively. The alkaline α-amylase AA11 was further characterized. At pH 9.0 the purified enzyme AA11 was highly stable while retaining 88-100% functional viability at temperature range from 35 to 65 °C, confirming its thermostability nature. It showed stability with powdered and liquid detergents at 7 mg/mL and 100-fold dilutions respectively. AA11 efficiently removed the starch stain from cotton fabrics. The findings of this study indicate that the isolate CH11 is a source of novel alkaline α-amylase that has promising application in food and detergent industries.

Isoleucine Regulates the Synthesis of Pancreatic Enzymes via the Activation of mRNA Expression and Phosphorylation in the Mammalian Target of Rapamycin Signalling Pathways in Pancreatic Tissues.

This study aimed to investigate the effects of isoleucine (Ile) on the synthesis and secretion of digestive enzymes and cellular signalling in the pancreatic tissue of dairy goats. The pancreatic tissues were incubated in buffer containing 0, 0.40, 0.80, and 1.60 mM Ile. High levels of Ile significantly increased the buffer release and total concentration of ɑ-amylase in the tissues (P < 0.001). The total trypsin and chymotrypsin concentrations in each of the Ile groups were significantly higher than those in the control group (P < 0.05); however, lipase was not affected. High levels of Ile significantly increased ɑ-amylase mRNA expression (P < 0.001) but had no effect on the mRNA expression of trypsin, chymotrypsin, or lipase. Ile did not affect S6K1 phosphorylation levels. High levels of Ile significantly increased the expression of the γ isoform of 4EBP1 (P < 0.001), which indicated that the phosphorylation of 4EBP1 was significantly increased. The phosphorylation level of eEF2 gradually decreased with the addition of Ile (P < 0.001). These results suggested that high doses of Ile can regulate the excretion of enzymes, especially ɑ-amylase, in the pancreatic tissues of dairy goats by modulating mTOR signalling, and this regulation is independent of the mTOR-S6K1 pathway.

Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes.

Aspergillus niger is an important industrial producer of enzymes due to its high capacity for producing exocellular secretory proteins. The CRISPR/Cas9 system has been developed as a genetic manipulation tool in A. niger. However, only the basic functions of the CRISPR/Cas9 system, such as codon optimization of Cas9 nucleases and promoter screening of guide RNA (gRNA) expression, have been developed in A. niger. The CRISPR/Cas9 system for manipulating large genomic fragments and multiple gene knock-ins still needs to be established. Here, we improved the CRISPR/Cas9 homologous direct repair (CRISPR-HDR) tool box based on donor DNAs (dDNAs) and plasmid harboring AMA1 and the pyrG marker, allowing recycling of pyrG and Cas9 components. Furthermore, we used the CRISPR-HDR tool box to knock out the 0 kb (protospacer only), 2 kb, 10 kb and even 50 kb gene fragments. This CRISPR-HDR tool box could also be used to simultaneously knock in multiple genes at the loci of two highly expressed extracellular secreted proteins, glucoamylase A (glaA) and alpha-amylase (amyA, two copies). In our study, two or three copies of glucose oxidase (goxC) were precisely knocked in at the loci of amyA and glaA, resulting in 4-fold increased enzyme activity (869.86 U/mL). This CRISPR-HDR tool box can be easily manipulated, and the AMA1-based plasmid can be easily removed under selective pressure of 5-fluoroorotic acid and uridine.

Proteases HtrA and HtrB for α-amylase secreted from Bacillus subtilis in secretion stress.

HtrA and HtrB are two important proteases across species. In biotechnological industries, they are related to degradation of secreted heterologous proteins from bacteria, especially in the case of overproduction of α-amylases in Bacillus subtilis. Induction of HtrA and HtrB synthesis follows the overproduction of α-amylases in B. subtilis. This is different from the order usually observed in B. subtilis, i.e., the production of proteases is prior to the secretion of proteins. This discrepancy suggests three possibilities: (i) HtrA and HtrB are constantly synthesized from the end of the exponential phase, and then are synthesized more abundantly due to secretion stress; (ii) There is a hysteresis mechanism that holds HtrA and HtrB back from their large amount of secretion before the overproduction of α-amylases; (iii) Heterologous amylases could be a stress to B. subtilis leading to a general response to stress. In this review, we analyze the literature to explore these three possibilities. The first possibility is attributed to the regulatory pathway of CssR-CssS. The second possibility is because sigma factor σD plays a role in the overproduction of α-amylases and is subpopulation dependent with the switch between "ON" and "OFF" states that is fundamental for a bistable system and a hysteresis mechanism. Thus, sigma factor σD helps to hold HtrA and HtrB back from massive secretion before the overproduction of α-amylases. The third possibility is that several sigma factors promote the secretion of proteases at the end of the exponential phase of growth under the condition that heterologous amylases are considered as a stress.

Mutations of Kluyveromyces lactis dolichol kinase enhances secretion of recombinant proteins.

Although there are similarities in the core steps of the secretion pathway from yeast to higher eukaryotes, significant functional differences exist even among diverse yeast species. Here, we used next-generation sequencing to identify two mutations in the Kluyveromyces lactis KlSEC59 gene, encoding dolichol kinase (DK), which are responsible for an enhanced secretion phenotype in a previously isolated mutant, MD2/1-9. Compared with the temperature-sensitive Saccharomyces cerevisiae sec59-1 mutant, which exhibits reduced N-glycosylation and decreased secretory efficacy, the identified K. lactis DK mutations had fewer effects on glycosylation, as well as on survival at high temperature and cell wall integrity. Moreover, despite some glycosylation defects, double DK mutations (G405S and I419S) in the K. lactis mutant strain demonstrated three times the level of recombinant α-amylase secretion as the wild-type strain. Overexpression of potential suppressors KlMNN10, KlSEL1, KlERG20, KlSRT1, KlRER2, KlCAX4, KlLPP1 and KlDPP1 in the DK-mutant strain restored carboxypeptidase Y glycosylation to different extents and, with the exception of KISRT1, reduced α-amylase secretion to levels observed in wild-type cells. Our results suggest that enhanced secretion related to reduced activity of mutant DK in K. lactis results from mild glycosylation changes that affect activity of other proteins in the secretory pathway.

High-level expression of a novel α-amylase from Thermomyces dupontii in Pichia pastoris and its application in maltose syrup production.

A novel α-amylase gene (TdAmyA) with an open reading frame of 1431 bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314 U/mL was obtained with protein content of 28.7 mg/mL after 168 h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2 kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60 °C and pH 6.5, and was thermostable up to 55 °C within pH 4.5-10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8 h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.

Leucine regulates α-amylase and trypsin synthesis in dairy calf pancreatic tissue in vitro via the mammalian target of rapamycin signalling pathway.

Starch digestion in the small intestines of the dairy cow is low, to a large extent, due to a shortage of syntheses of α-amylase. One strategy to improve the situation is to enhance the synthesis of α-amylase. The mammalian target of rapamycin (mTOR) signalling pathway, which acts as a central regulator of protein synthesis, can be activated by leucine. Our objectives were to investigate the effects of leucine on the mTOR signalling pathway and to define the associations between these signalling activities and the synthesis of pancreatic enzymes using an in vitro model of cultured Holstein dairy calf pancreatic tissue. The pancreatic tissue was incubated in culture medium containing l-leucine for 3 h, and samples were collected hourly, with the control being included but not containing l-leucine. The leucine supplementation increased α-amylase and trypsin activities and the messenger RNA expression of their coding genes (P <0.05), and it enhanced the mTOR synthesis and the phosphorylation of mTOR, ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1 (P <0.05). In addition, rapamycin inhibited the mTOR signal pathway factors during leucine treatment. In sum, the leucine regulates α-amylase and trypsin synthesis in dairy calves through the regulation of the mTOR signal pathways.