Development of bacteria-based bioorganic phosphate fertilizer enriched with rock phosphate for sustainable wheat production.

Phosphorous (P) is a limiting macronutrient for crop growth. Its deficiency prevents plant development leading to an extensive use of phosphatic fertilizers globally. Bio-organic phosphate (BOP) fertilizer provides a sustainable approach to optimize nutrient availability, enhance crop yield, and mitigate the negative impacts of chemical fertilizers on the environment. Therefore, the present study integrates the application of heat-tolerant phosphate-solubilizing bacteria, rock phosphate, and organic materials for the development of BOP. For this purpose, potential heat-tolerant phosphate-solubilizing bacteria (PSB) were isolated from major wheat-growing areas of southern Punjab. Five isolates were the efficient phosphate solubilizers based on in vitro phosphate-solubilizing activity (291-454 µg ml-1 and 278-421 µg ml-1) with a concomitant decrease in pH (up to 4.5) at 45°C and 50°C, respectively. These PSB were used for the development of potential consortia that are compatible and showed high P solubilization. In planta evaluation of these PSB consortia in a pot experiment under net house conditions showed that consortium-2 had a favorable impact on growth parameter with enhanced grain yield (9.63 g plant-1) and soil available P (10 µg g-1) as compared with 80% uninoculated control. The microcosm study was conducted to evaluate PSB consortium-2 integrated with carrier material (plant material and filter mud) and rock phosphate as BOP increased total phosphorous (14%) as compared with uninoculated controls. Plant-based BOP showed higher viable count (3.5 × 108\u00B0CFU) as compared with filter mud-based BOP. Furthermore, the effect of BOP on wheat growth parameters revealed that BOP showed a promising influence on grain yield (4.5 g plant-1) and soil available P (10.7 µg g-1) as compared with uninoculated 80 and 100% controls. Principle component analysis (PCA) further validates a positive correlation between BOP with grain weight and plant height and soil available P as compared with both 80 and 100% controls. For the first time, this study reports the combined application of bio-organic phosphate fertilizer and heat-tolerant PSB, which offers an eco-friendly option to harvest better wheat yield with low fertilizer input.

Bioactive Streptomycetes: A Powerful Tool to Synthesize Diverse Nanoparticles With Multifarious Properties.

Nanobiotechnology has gained significant attention due to its capacity to generate substantial benefits through the integration of microbial biotechnology and nanotechnology. Among microbial organisms, Actinomycetes, particularly the prominent genus Streptomycetes, have garnered attention for their prolific production of antibiotics. Streptomycetes have emerged as pivotal contributors to the discovery of a substantial number of antibiotics and play a dominant role in combating infectious diseases on a global scale. Despite the noteworthy progress achieved through the development and utilization of antibiotics to combat infectious pathogens, the prevalence of infectious diseases remains a prominent cause of mortality worldwide, particularly among the elderly and children. The emergence of antibiotic resistance among pathogens has diminished the efficacy of antibiotics in recent decades. Nevertheless, Streptomycetes continue to demonstrate their potential by producing bioactive metabolites for the synthesis of nanoparticles. Streptomycetes are instrumental in producing nanoparticles with diverse bioactive characteristics, including antiviral, antibacterial, antifungal, antioxidant, and antitumor properties. Biologically synthesized nanoparticles have exhibited a meaningful reduction in the impact of antibiotic resistance, providing resources for the development of new and effective drugs. This review succinctly outlines the significant applications of Streptomycetes as a crucial element in nanoparticle synthesis, showcasing their potential for diverse and enhanced beneficial applications.

Draft genome sequence of extracellular enzyme-producing Bacillus paralicheniformis strain NBG-07.

The genome of Bacillus paralicheniformis strain NBG-07 was sequenced using Illumina sequencing due to its ability to produce thermostable enzymes of industrial importance. The strain was isolated from the soil. Annotation of the draft genome revealed genes involved in the production of different enzymes, including alpha-amylase, protease, cellulase, and laccase.

Surfactin-Conjugated Silver Nanoparticles as an Antibacterial and Antibiofilm Agent against Pseudomonas aeruginosa.

The emergence of antimicrobial resistance is an alarming global health concern and has stimulated the development of novel functional nanomaterials to combat multi-drug-resistant (MDR) bacteria. In this work, we demonstrate for the first time the synthesis and application of surfactin-coated silver nanoparticles as an efficient antibacterial and antibiofilm agent against the drug-resistant bacteria Pseudomonas aeruginosa for safe dermal applications. Our in vivo studies showed no significant superficial dermal irritation, edema, and erythema, while microscopic analysis revealed that surfactin-coated silver nanoparticles caused no pathological alterations at the applied concentrations. These results support the potential use of surfactin-coated silver nanoparticles against drug-resistant bacterial biofilm infections and in skin wound dressing applications.

Dietary Supplementation of Microbial Dextran and Inulin Exerts Hypocholesterolemic Effects and Modulates Gut Microbiota in BALB/c Mice Models.

Microbial exopolysaccharides (EPSs), having great structural diversity, have gained tremendous interest for their prebiotic effects. In the present study, mice models were used to investigate if microbial dextran and inulin-type EPSs could also play role in the modulation of microbiomics and metabolomics by improving certain biochemical parameters, such as blood cholesterol and glucose levels and weight gain. Feeding the mice for 21 days on EPS-supplemented feed resulted in only 7.6 ± 0.8% weight gain in the inulin-fed mice group, while the dextran-fed group also showed a low weight gain trend as compared to the control group. Blood glucose levels of the dextran- and inulin-fed groups did not change significantly in comparison with the control where it increased by 22 ± 5%. Moreover, the dextran and inulin exerted pronounced hypocholesterolemic effects by reducing the serum cholesterol levels by 23% and 13%, respectively. The control group was found to be mainly populated with Enterococcus faecalis, Staphylococcus gallinarum, Mammaliicoccus lentus and Klebsiella aerogenes. The colonization of E. faecalis was inhibited by 59-65% while the intestinal release of Escherichia fergusonii was increased by 85-95% in the EPS-supplemented groups, respectively, along with the complete inhibition of growth of other enteropathogens. Additionally, higher populations of lactic acid bacteria were detected in the intestine of EPS-fed mice as compared to controls.

Surface-enhanced Raman spectroscopy for monitoring antibacterial activity of imidazole derivative (1-benzyl-3-(sec­butyl)-1H-imidazole-3-ium bromide) against Bacillus subtilis and Escherichia coli.

BACKGROUND: Bacterial resistance against antibiotics remains a challenge and Raman Spectroscopy (SERS) may provide critical information concerning this. OBJECTIVES: In the current study, surface enhances Raman spectroscopy (SERS) has been used to determine the biochemical changes induced during the antibacterial activity of the in house synthesized imidazole derivative (1-benzyl-3-(sec­butyl)-1H-imidazole-3-ium bromide) in comparison to commercially available drugs (fasygien) against both gram-positive and gram-negative bacteria. METHODS: For this purpose, the antibacterial activity of this compound was assessed on Bacillus subtilis and Escherichia coli. The SERS spectral changes are detected which can be associated with the biochemical changes in the bacterial cells as a result of the application of both drugs, including fasygien and the imidazole derivative drug demonstrating the technique's potential for analyzing the antibacterial activities of drug candidates. RESULTS: The chemometric techniques such as Principal Component Analysis (PCA) and Partial Least Squares-Discriminant Analysis (PLS-DA) were performed for the differentiation of SERS spectral data sets of unexposed, exposed with imidazole derivative and commercially available antibacterial drugs for two different bacteria including E. coli and Bacillus. CONCLUSIONS: PCA was found helpful for the qualitative differentiation of all drug-treated E. coli and Bacillus in the form of separate clusters of spectral data sets and PLS-DA discriminated the unexposed and the exposed bacteria with imidazole derivative and commercially available drug with 93% sensitivity and 96% specificity for Bacillus and with 90% sensitivity and 89% specificity for E. coli.

Mitigation of water scarcity with sustained growth of Rice by plant growth promoting bacteria.

Climate change augments the risk to food security by inducing drought stress and a drastic decline in global rice production. Plant growth-promoting bacteria (PGPB) have been known to improve plant growth under drought stress. Here in the present study, we isolated, identified, and well-characterized eight drought-tolerant bacteria from the rice rhizosphere that are tolerant to 20% PEG-8000. These strains exhibited multiple plant growth-promoting traits, i.e., 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, exopolysaccharide production, phosphate (P)-solubilizing activity (51-356 µg ml-1), indole-3 acetic acid (IAA) production (14.3-46.2 µg ml-1), and production of organic acids (72-178 µg ml-1). Inoculation of bacterial consortium (Bacillus subtilis NM-2, Brucella haematophilum NM-4, and Bacillus cereus NM-6) significantly improved seedling growth and vigor index (1009.2-1100) as compared to non-inoculated stressed plants (630-957). Through rhizoscanning, efficiency of the consortium was validated by improved root parameters such as root length (17%), diameter, and surface area (18%) of all tested genotypes as compared with respective non-inoculated stressed treatments. Furthermore, the response of consortium inoculation on three rice genotypes was positively correlated with improved plant growth and drought stress ameliorating traits by the accumulation of osmoprotectant, i.e., proline (85.8%-122%), relative water content (51%), membrane stability index (64%), and production of antioxidant enzymes to reduce oxidative damage by reactive oxygen species. A decrease in temperature and improved chlorophyll content of inoculated plants were found using infrared thermal imaging and soil plant analyzer development (SPAD), respectively. The key supporting role of inoculation toward stress responses was validated using robust techniques like infrared thermal imaging and an infrared gas analyzer. Furthermore, principal component analysis depicts the contribution of inoculation on stress responses and yield of tested rice genotypes under water stress. The integration of drought-tolerant rice genotype (NIBGE-DT02) and potential bacterial strains, i.e., NM-2, NM-4, and NM-6, can serve as an effective bioinoculant to cope with water scarcity under current alarming issues related to food security in fluctuating climate.

Genome analysis of novel Apilactobacillus sp. isolate from butterfly (Pieris canidia) gut reveals occurrence of unique glucanogenic traits and probiotic potential.

This study was conducted with a perception that fructose-rich niches may inhabit novel species of lactic acid bacteria that are gaining importance as probiotics and for the production of exopolysaccharides that have applications in food and pharmaceuticals. Recently, some Lactobacillus species have been reclassified as fructophilic lactic acid bacteria due to their preference for fructose over glucose as a carbon source. These bacteria are likely to be found in fructose rich niches such as flower nectar and insects that feed on it. We explored the butterfly gut and acquired a new isolate, designated as F1, of fructophilic lactic acid bacteria, which produces a glucan-type exopolysaccharide. Whole genome sequencing and in silico analysis revealed that F1 has significantly lower average nucleotide identity and DNA-DNA hybridization values as compared to its closest Apilactobacillus neighbors in phylogenetic analysis. Therefore, we declare the isolate F1 as a novel Apilactobacillus species with the proposed name of Apilactobacillus iqraium F1. Genome mining further revealed that F1 harbors genes for exopolysaccharide synthesis and health-promoting attributes. To this end, F1 is the only Apilactobacillus species harboring three diverse α-glucan-synthesis genes that cluster with different types of dextransucrases in the dendrogram. Moreover, many nutritional marker genes, as well as genes for epithelial cell adhesion and antimicrobial synthesis, were also detected suggesting the probiotic attributes of F1. Overall analysis suggests A. iqraium sp. F1 be a potential candidate for various health beneficial and pharmaceutical applications.

Draft Genome Sequences of Bacillus velezensis Strains AF_3B and OS2, Bacillus amyloliquefaciens Strain BS9, Bacillus halotolerans Strain A1, and Bacillus sp. Strain BS3, Producing Biosurfactants with Antimicrobial Potential.

Five environmental Bacillus strains were sequenced, of which three were isolated from the rhizosphere of agricultural soil and one each from Attock Oil Refinery and Khewra Salt Mine in Pakistan. The strains can be used for plant growth promotion and biosurfactant activity brought about by secondary metabolites.

Revealing Genome-Based Biosynthetic Potential of Streptomyces sp. BR123 Isolated from Sunflower Rhizosphere with Broad Spectrum Antimicrobial Activity.

Actinomycetes, most notably the genus Streptomyces, have great importance due to their role in the discovery of new natural products, especially for finding antimicrobial secondary metabolites that are useful in the medicinal science and biotechnology industries. In the current study, a genome-based evaluation of Streptomyces sp. isolate BR123 was analyzed to determine its biosynthetic potential, based on its in vitro antimicrobial activity against a broad range of microbial pathogens, including gram-positive and gram-negative bacteria and fungi. A draft genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 was attained, containing a GC content of 72.63% and 8103 protein coding genes. Many antimicrobial, antiparasitic, and anticancerous compounds were detected by the presence of multiple biosynthetic gene clusters, which was predicted by in silico analysis. A novel metabolite with a molecular mass of 1271.7773 in positive ion mode was detected through a high-performance liquid chromatography linked with mass spectrometry (HPLC-MS) analysis. In addition, another compound, meridamycin, was also identified through a HPLC-MS analysis. The current study reveals the biosynthetic potential of Streptomyces sp. isolate BR123, with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study compared the isolate BR123 with other Streptomyces strains, which may expand the knowledge concerning the mechanism involved in novel antimicrobial metabolite synthesis.

Draft Genome Sequence of Streptomyces sp. Strain R1, Isolated from Water Canal Sediments, Possessing Antimicrobial and Plant Growth Promoting Capabilities.

We present the genome sequence of Streptomyces sp. strain R1, isolated from water canal sediments and possessing genes responsible for antimicrobial metabolites and plant growth promotion. The genome assembly contains 7,936,694 bp with 72.24% of guanine-cytosine content. This genome will provide basic knowledge of the genes and pathways involved in the above mechanisms.

Production of a high molecular weight levan by Bacillus paralicheniformis, an industrially and agriculturally important isolate from the buffalo grass rhizosphere.

A new exopolysaccharide (EPS) producing Gram-positive bacterium was isolated from the rhizosphere of Bouteloua dactyloides (buffalo grass) and its EPS product was structurally characterized. The isolate, designated as LB1-1A, was identified as Bacillus paralicheniformis based on 16S rRNA gene sequence and phylogenetic tree analysis. The EPS produced by LB1-1A was identified as a levan, having ß(2 → 6) linked backbone with ß(2 → 1) linkages at the branch points (4.66%). The isolate LB1-1A yielded large amount (~ 42 g/l) of levan having high weight average molecular weight (Mw) of 5.517 × 107 Da. The relatively low degree of branching and high molecular weight of this levan makes B. paralicheniformis LB1-1A a promising candidate for industrial applications.

Biodegradation and Subsequent Toxicity Reduction of Co-contaminants Tribenuron Methyl and Metsulfuron Methyl by a Bacterial Consortium B2R.

AllyMax is a widely used herbicide formulation in wheat-rice cropping areas of the world. The residues of its active ingredients, tribenuron methyl (TBM) and metsulfuron methyl (MET), persist in soil and water as co-contaminants, and cause serious threats to nontarget organisms. This study was performed to assess the potential of a bacterial consortium for the degradation and detoxification of TBM and MET individually and as co-contaminants. A bacterial consortium (B2R), comprising Bacillus cereus SU-1, Bacillus velezensis OS-2, and Rhodococcus rhodochrous AQ1, capable of degrading TBM and MET in liquid cultures was developed. Biodegradation of TBM and MET was optimized using the Taguchi design of experiment. Optimum degradation of both TBM and MET was obtained at pH 7 and 37 °C. Regarding media composition, optimum degradation of TBM and MET was obtained in minimal salt medium (MSM) supplemented with glucose, and MSM without glucose, respectively. The consortium simultaneously degraded TBM and MET (94.8 and 80.4%, respectively) in cultures containing the formulation AllyMax, where TBM and MET existed as co-contaminants at 2.5 mg/L each. Mass spectrometry analysis confirmed that during biodegradation, TBM and MET were metabolized into simpler compounds. Onion (Allium cepa) root inhibition and Comet assays revealed that the bacterial consortium B2R detoxified TBM and MET separately and as co-contaminants. The consortium B2R can potentially be used for the remediation of soil and water co-contaminated with TBM and MET.

Whole Genome Sequence Analysis of a Novel Apilactobacillus Species from Giant Honeybee (Apis dorsata) Gut Reveals Occurrence of Genetic Elements Coding Prebiotic and Probiotic Traits.

Apilactobacillus spp. are classified as obligate fructophilic lactic acid bacteria (FLAB) that inhabit fructose-rich niches such as honeybee gut. Lactic acid bacteria are an important component of the gut microbiome and play a crucial role in maintaining gut health. In this study, a new FLAB strain HBW1, capable of producing glucan-type exopolysaccharide, was isolated from giant honeybee (Apis dorsata) gut and subjected to whole genome sequencing (WHS) to determine its health-beneficial traits. The genome size of the isolate was 1.49 Mb with a GC content of 37.2%. For species level identity, 16S rDNA sequence similarity, genome to genome distance calculator (dDDH), and average nucleotide identity (ANI) values were calculated. Phylogenetic analysis showed that the isolate HBW1 belongs to the Apilactobacillus genus. The dDDH and ANI values in comparison with closely clustered Apilactobacillus kunkeei species were 52% and 93.10%, respectively. Based on these values, we concluded that HBW1 is a novel species of Apilactobacillus, and we propose the name Apilactobacillus waqarii HBW1 for it. Further, WHS data mining of HBW1 revealed that it harbors two glucosyltransferase genes for prebiotic glucan-type exopolysaccharide synthesis. Moreover, chaperon (clp) and methionine sulfoxide reductase (msrA, msrB, and msrC) genes as well as nutritional marker genes for folic acid (folD) and riboflavin biosynthesis (rib operon), important for conferring probiotic properties, were also detected. Occurrence of these genetic traits make HBW1 an excellent candidate for application to improve gut function.

Production of bimodal molecular weight levan by a Lactobacillus reuteri isolate from fish gut.

An exopolysaccharide (EPS) synthesizing potentially probiotic Gram-positive bacterial strain was isolated from fish (Tor putitora) gut, and its EPS was structurally characterized. The isolate, designated as FW2, was identified as Lactobacillus reuteri through 16S rRNA gene sequencing and phylogenetic analysis. This isolate produces fructan-type EPS using sucrose as a substrate. Based on 13C-NMR spectroscopy, methylation analysis and monosaccharide composition, the EPS was identified as a linear levan polymer with fructose as main constituent linked via ß(2 → 6) linkages. Based on molecular weight (MW) distribution, two groups of levan were found to be produced by the isolate FW2: one with high MW (4.6 × 106 Da) and the other having much lower MW (1.2 × 104 Da). The isolate yielded about 14 g/L levan under optimized culturing parameters including aeration conditions, pH, temperature and substrate concentration. The obtained bimodal molecular weight linear levan is the first of its type to be synthesized by a L. reuteri isolate from fish gut. Bimodal molecular weight prebiotic levan together with the probiotic potential of the producing strain would provide a new promising synbiotic combination for use in aqua culture.

Production of a broad spectrum streptothricin like antibiotic from halotolerant Streptomyces fimbriatus isolate G1 associated with marine sediments.

Streptomyces have been reported as a remarkable source for bioactive secondary metabolites with complex structural and functional diversity. In this study, 35 isolates of genus Streptomyces were purified from rhizospheric and marine soils collected from previously unexplored habitats and screened for antimicrobial activities. One of these isolates, G1, when tested in vitro, was found highly active against wide range of microbes including Gram-positive, Gram-negative bacteria, and different fungal pathogens. It was identified as mesophilic, alkaliphilic, and moderately halotolerant as it showed optimum growth at temperature 30 °C, pH 8.0 in casein-starch-peptone-yeast extract-malt extract medium supplemented with 5% NaCl. Sequence analysis of the 16S rRNA gene indicated 100% identity of this isolate to Streptomyces fimbriatus. Moreover, maximum antimicrobial activity was achieved in starch nitrate medium supplemented with 1% glycerol as carbon and 0.03% soy meal as nitrogen source. The antimicrobial compounds produced by this isolate were extracted in methanol. Bioassay-guided fractionation through thin layer chromatography of methanolic extract resulted in the separation of a most active fraction with an Rf value of 0.46. This active fraction was characterized by FTIR and LCMS analysis and found similar to streptothricin D like antibiotic with m/z 758.42.

Draft Genome Sequence of Streptomyces sp. Strain BR123, Endowed with Broad-Spectrum Antimicrobial Potential.

The genome of Streptomyces sp. strain BR123, isolated from rhizospheric soil that exhibited promising antimicrobial properties, was sequenced and assembled. Here, we report an 8,157,040-bp genome sequence with a G+C content of 72.63%. This genome sequence enlightens the genes responsible for the production of secondary metabolites and antimicrobial compounds by this strain.

Growth Inhibition of Common Enteric Pathogens in the Intestine of Broilers by Microbially Produced Dextran and Levan Exopolysaccharides.

Antibiotics are generally applied for treatment or as subtherapeutic agents to overcome diseases caused by pathogenic bacteria including Escherichia coli, Salmonella and Enterococcus species in poultry. However, due to their possible adverse effects on animal health and to maintain food safety, probiotics, prebiotics, and synbiotics have been proposed as alternatives to antibiotic growth promoters (AGPs) in poultry production. In this study, the effects of prebiotics on the augmentation of broiler's indigenous gut microbiology were studied. Day old 180 broilers chicks were divided into four treatment groups: G, L, C1, and C2. The groups G and L were fed with basal diet containing 3% dextran and 3% levan, respectively. Control groups were fed with basal diets without antibiotic (C1) and with antibiotics (C2). The experimental groups showed decreased mortality as compared to control groups. After 35 days, the chickens were euthanized and intestinal fluid was analyzed for enteric pathogens on chromogenic agar plates and by 16S rRNA gene sequencing. Inhibition of the growth of E. coli and Enterococcus was observed in groups G and L, respectively, whereas Salmonella was only present in group C1. Also, high populations of lactic acid bacteria were detected in the intestine of prebiotic fed birds as compared to controls. These results depict that dextran and levan have the potential to replace the use of antibiotics in poultry feed for inhibiting the growth of common enteric pathogens. To the best of our knowledge, this is the first study where effects of dextran and levan on intestinal microbiota of broilers have been reported.

Fungal strains isolation, identification and application for the recovery of Zn(II) ions.

Fungal biomass proves to be highly efficient for the treatment of wastewater as well as recovery of metal ions from wastewater. Present investigation was aimed to evaluate the efficiency of indigenous fungal isolates for the sequestration of Zn(II) ions aqueous solution. Among twenty five fungal isolates, Aspergillus oryzae SV/09 (AO SV/09), Aspergillus flavus NA9 (AF NA9) and Paecilomyces formosus DTO 63f4 (PF DTO-63f4) were identified by gene sequencing of ITS regions of the ribosomal DNA (rDNA). The AO SV/09, AF NA9 and PF DTO-63f4 showed promising efficiency for the biosorption of Zn(II) ions. Zn(II) ions adsorption was endothermic in nature and data fitted will to the Freundlich isotherm with correlation coefficients values of 0.99, 0.98 and 0.99 for AO SV/09, AF NA9 and PF DTO-63f4, respectively. Pseudo-second order kinetic model explained well the Zn(II) adsorption kinetic of Zn(II) ions onto biosorbents. The adsorbed Zn(II) ions were desorbed using HCl and 85.5, 75.3, 73.7 (%) Zn(II) ions were recovered from AO SV/09, AF NA9 and PF DTO-63f4 sorbents, respectively. The fungal biosorbents were successfully recycled up to five cycles. Based on sorption, recovery and regeneration, the application of fungal bio-sorbents for the sequestration and recovery of Zn(II) ions is suggested from wastewater and could possibly be extended for the recovery of other heavy metal ions from wastewater.

Solid State Fermentation of a Raw Starch Digesting Alkaline Alpha-Amylase from Bacillus licheniformis RT7PE1 and Its Characteristics.

The thermodynamic and kinetic properties of solids state raw starch digesting alpha amylase from newly isolated Bacillus licheniformis RT7PE1 strain were studied. The kinetic values Q p , Y p/s , Y p/X , and q p were proved to be best with 15% wheat bran. The molecular weight of purified enzyme was 112 kDa. The apparent K m and V max values for starch were 3.4 mg mL(-1) and 19.5 IU mg(-1) protein, respectively. The optimum temperature and pH for α -amylase were 55°C, 9.8. The half-life of enzyme at 95°C was 17h. The activation and denaturation activation energies were 45.2 and 41.2 kJ mol(-1), respectively. Both enthalpies (ΔH (∗)) and entropies of activation (ΔS (∗)) for denaturation of α -amylase were lower than those reported for other thermostable α -amylases.