Whole-genome sequence and assembly of the sporogenic Bacillus paralicheniformis T7 strain with high proteolytic and amylolytic activities

The varied diverse enzymatic characteristics of Bacillus species render them advantageous for several applications, including food processing, agriculture, biomedicine, biofuel generation, medicines, hydrolysis, bioremediation, and natural polymer processing (Muras et al., 2021;Sharma et al., 2021;Zhao et al., 2021). Several bacillary strains exhibit elevated production capabilities (Contesini et al., 2018), with B. paralicheniformis strains being influential prominent in the enzyme manufacturing sector. For instance, B. paralicheniformis MKU3 is recognized for its ability to production ofe proteases that effectively breaks down feather keratin (Santha Kalaikumari et al., 2019), while B. paralicheniformis BL.HK produces extracellular proteases utilized used in the enzymatic processing of animal hides (Akhtar et al., 2024). Furthermore, B. paralicheniformis HR-1 and B. haynesii HR-5, isolated from bottom sediments, are utilized employed for the production of alkaline proteases applicable in the textile and leather industries (Thakor et al., 2025). Proteases obtained from B. paralicheniformis T7 also exhibit significant keratinase activity in the hydrolysis of avian feathers, wool, horns, hooves, and hides (Aktayeva and Khassenov, 2024b;Aktayeva and Khassenov, 2024a). Alongside proteases and keratinases, B. paralicheniformis strains produce α-amylases (Božić et al., 2020), phosphatases (Abdelgalil et al., 2021), esterases (Ganesh Kumar et al., 2021), andxylanases (Ngom et al., 2023) alongside proteases and keratinases. Specifically, the α-amylase produced by B. paralicheniformis ATCC 9945a effectively produces α-amylase, which hydrolyzes starch effectively without pretreatment (Božić et al., 2020). Additionally, the α-amylase from B. paralicheniformis GRA2 was applied integrated into a multienzyme preparation for the hydrolysis of food waste (Roslan et al., 2021).The manuscript (from Introduction to the end of Limitations) has been edited for language, grammar, clarity, and consistency. Comments have also been left where any additional input is required. Please note that US English has been used throughout the manuscript. As requested, no journal formatting has been undertaken. assessed according to (Coêlho et al., . (2016) using azocasein (Sigma-Aldrich) as a substrate in 50 mM Tris-HCl (pH 9.0) at 60 °С. Collagenase activity was determined evaluated according to (Chavira et al., . (1984)) with using azocoll (Sigma-Aldrich) as a substrate in 50 mM Tris-HCl (pH 9.0) supplemented with 1 mM CaCl2 at 37 °C. Milk clotting activity was determined measured according following the method ofto ( Akishev et al., . (2022) with using cow milk as a substrate.Determination of αAlpha-amylase activity was performed determined in 100 mM phosphate buffer, pH 6.0, at 85 °C C by via the method of reducing sugars sugar method adapted using to potato starch as the substrate (Sigma-Aldrich) as a substrate (Kiribayeva et al., 2024). Esterase activity was determined ascertained at 40 °C C in 50 mM phosphate buffer, pH 7.0, at 40 C according to (Zhao et al., . (2022) by means ofemploying 4-nitrophenyl acetate (Thermo Scientific, Waltham, MA, USA) or 4-nitrophenyl octanoate (Thermo Fisher, Kandel, Germany) as a substrate.Alkaline phosphatase activity was assayed according tofollowing the method of ( Abdelgalil et al., . (2021) using p-nitrophenyl phosphate disodium salt 6-hexahydrate (PanReacAppliChem, Darmstadt, Germany) as a substrate in 100 mM phosphate buffer, pH 10.3, at 70 °C. C. Phytase activity was assayed assessed according toas described by ( Choi et al., . (2001) using phytic acid sodium salt as a substrate in 100 mM Tris-HCl (pH 8.0) at 60 °CC. All experiments were performed in triplicate. Enzymatic activity measurement data were obtained derived through from independent activity assays, with mean values, standard deviations (SD), and p-values calculated using GraphPad Prism version 8.0.1 (GraphPad Software, La Jolla, CA, USA, www.graphpad.com). All data are presented as the means ± SD (n = 3).Endospores were obtained generated by culturing the strain on Arret-Kirshbaum sporulation agar or Modified modified Nutrient nutrient agar at 37 °C, and in Difco sporulation medium in in a shaker shaking incubator (150 rpm) at 37 °C and 150 rpm. Cultivation time was determined by microbiological observation using Schaeffer-Fulton staining. Cultivation was stopped when the spore-to-cell ratio reached 1:1 or until when spores predominated. Cells and spores were collected harvested by centrifugation; . to To remove the remaining vegetative cells, the suspension was heated for 20 minutes at 90 °C.The strain''s resistance to antibiotics resistance profile was tested by using the disk diffusion test method according toas described by (Matuschek et al., . (2014). The following antibiotics were used: ampicillin (TM Media, 10 µg/disc, Lot # 041210), chloramphenicol (TM Media, 30 µg/disc, Lot # 0712109), ciprofloxacin (TM Media, 5 µg/disc, Lot # 0812110), clindamycin (TM Media, 2 µg/disc, Lot # 0012109), erythromycin (TM Media, 15 µg/disc, Lot # 0412109), gentamicin (TM Media, 120 µg/disc, Lot # 0012012), kanamycin (TM Media, 30 µg/disc, Lot # 0612110), nalidixic acid (TM Media, 30 µg/disc, Lot # 0922108), penicillin-G (TM Media, 10 µg/disc, Lot # 0422108), rifampicin (TM Media, 5 µg/disc, Lot # 0712109), streptomycin (TM Media, 10 µg/disc, Lot # 0912109), tetracycline (TM Media, 30 µg/disc, Lot # 0012109), tobramycin (TM Media, 10 µg/disc, Lot # 1212109), and cefazolin (HiMedia, 30 µg/disc, Lot # 0000156850).The culture Ssupernatant after cultivation of the strain was used for zymography zymographic and mass spectrometry spectrometric analysis. Zymography Zymographic analysis отформатировано: подстрочные отформатировано: Шрифт: курсив Добавлено примечание ([A4]): This heading was edited to reflect the information in the paragraph. Please ensure that this change is appropriate. was performed accordingly to (Aktayeva et al., . (2022) with using casein, keratin, and gelatin as substrates. To determine the specific protease classes, tThe protease following inhibitors were employed: phenylmethylsulfonyl fluoride, EDTA, E64, and Pepstatin A for protease class definition. Mass spectrometry spectrometric analysis of the secreted proteome was performed with using a Maxis Impact II Instrument (Bruker, Germany). The strain extracellular enzymes were identified on via the Mascot platform.The strain culture was cultivated in 10 mL of Nutrient nutrient broth. The cells were harvested by centrifugation at 6000 × g for 7 min at 4 °C. Genomic DNA was isolated using with a Genomic Wizard Purification Kit (Promega, Madison, WI, USA) following the manufacturer''s protocol. To determine tThe amount quantity and quality of the DNA were determined using, a NanoDrop OneC spectrophotometer (Thermo Scientific, Waltham, MA, USA) and agarose gel electrophoresis were used. The concentration obtained was 1.79 ng/µL, which corresponds to the a yield of 38 μg for of genomic DNA. Whole-genome libraries were generated using the Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit (SQK-LSK109) sequencing kit (Oxford Nanopore Technologies (ONT)), which. This process entailed the attaching ligation of sequencing adapters using a Ligation Sequencing Kit (SQK-LSK109) following the manufacturer's protocolretrieved from the website (https://nanoporetech.com/document/gDNA-sqk-lsk109). The generated libraries were quantified using a Qubit 2.0 fluorometer device (Invitrogen).Subsequently, the libraries were and sequenced on the MinION platform using a FLO-MIN106 (R9.4.1) flow cell. The unprocessedRaw data underwent base calling using Guppy v3.4.1,.Thereafter, followed by the removal of low-quality reads were filtered out to ensure high-quality data.. After Sequencing generated a cumulative count of 149,880 sequencing reads averaging 11.98 read quality and with a median read length of 5,597 base pairs and an average read quality score of 11.98, we acquired a cumulative count of 149,880 sequencing reads. The Epi2me pipeline, including the ""epi2me-labs/wf-bacterial-genomes" " module, was employed for additional downstream analysis. De novo genomeand construction. assembly was performed using Tthe Flye v.2.9.1-b1780 algorithm (Kolmogorov et al., 2019) algorithm was used to assemble the de novo genome. A single circular contig with 45.93% G + C content was generated, measuring of 4,360,494 bp with a G + C content of 45.93% andin length and exhibiting a high mean contig coverage of 360X was generated (Supplementary File 1, Table S1, Table S2). Prior toBefore annotation, the assembly was polished using Medaka (v.1.7.2) based on the same ONT reads.Genomic features were visualized via DNA Features Viewer. was used for visualization of annotated genomic features. Additional genome annotation and circular genome visualization (Figure 1) were performed using the Proksee (Grant et al., 2023). The PATRIC resource was accessed via the BV-BRC platform, which integrates and extends the former PATRIC database (Wattam et al., 2017;Olson et al., 2023). Genome completeness was assessed using BUSCO v6.0.0 (Manni et al., 2021) with against the bacillus_odb12 database using BUSCO v.6.0.0 (Manni et al., 2021). The assembly results showed that the assembly contained 97.2% complete BUSCOs (97.0% single-copy, 0.1% duplicated), 1.8% fragmented BUSCOs, and 1.0% missing BUSCOs (Supplementary File 1, Table S3). Additional CheckM2 tool has been used for assessment of genome The quality of the assembled Bacillus paralicheniformis T7 strain genome was further отформатировано: Шрифт: курсив отформатировано: Шрифт: курсив отформатировано: Шрифт: курсив verified using the CheckM2 tool (Supplementary File 1, Table S4) (Chklovski et al., 2023). The pPhylogenetic analysis was performed using CSI Phylogeny v.1.4 (Call SNPs & Infer Phylogeny) (Kaas et al., 2014) via the web service provided by the Center for Genomic Epidemiology (CGE) web service. As input, genome assemblies (FASTA format) of the studied strain and selected reference genomes were analyzed using the a reference-based SNP-calling workflow implemented in CSI Phylogeny, with the Bacillus paralicheniformis genome used serving as the reference.Default alignment and SNP-calling settings were applied. SNPs were filtered using the following parameters:based on a minimum depth at SNP positions of 10×, a minimum relative depth of 10%, a minimum distance between SNPs (pruning) of 10 bp, a minimum SNP quality score of 30, a minimum mapping quality of 25, and a minimum Z-score of 1.96. ----High-confidence SNPs that passed all filters were concatenated into a single alignment, and a maximum-likelihood phylogenetic tree was inferred using FastTree as implemented in CSI Phylogeny. Branch support values were estimated using the Shimodaira-Hasegawa-like (SH-like) approximate likelihood ratio test. The resulting phylogenetic tree was visualized and annotated using iTOL v.6 (Letunic and Bork, 2024).The present study provides the genetic sequence of strain T7, which was isolated from Kazakhastan soil in Kazakhstan. The bacterial cells of the strain were are Gram-positive, rodshaped, motile bacteria, and capable of sporulation. Bacterial cells occur both singly and in chains.When cultured on LB agar at 37 °C for 16 h, the strain forms milky, irregular colonies (2-4 mm in diameter)that are irregular in shape with cloudy and opaque areas, milky in color, 2-4 mm in diameter, characterized by with a glossy surface and cloudy, opaque regions. When cultured in Nutrient nutrient broth under aerobic conditions at 37 °C and shakenwith shaking at (150 rpm), the strain shows profuse growth within 24 h. During growth, the medium becomes cloudy and acquires a characteristic odor, and bacterial flakes form in the culture medium. A difficult-to-break film forms at the liquid-air interface. Based on these characteristics, the strain was classified as belonging to the genus Bacillus. Based on the resultsSequencing of the 16S rRNA genefragment sequencing, identified the strain was identified as Bacillus paralicheniformis (100% identity). B. paralicheniformis T7 grew well in both nutrient and lysogeny broths. Upon When cultivatedion on milk, keratin, and gelatin agar, the strain exhibited clearance zones appeared, indicative of its protease, keratinase, and gelatinase activities. The strain grows in across a wide range of temperatures range ranging fromof 30 to 55 °C and a pH range of 6.0-8.5. Phenotypic These phenotypic and biochemical characteristics of strain T7 demonstrate are typical features of Bacillus species and are similar toconsistent with those of other B. paralicheniformis species (Dunlap et al., 2015). When cultivated in a bioreactor, B. paralicheniformis T7 reaches achieves maximum proteolytic activity within 24 hours, which is less shorter than that reported offor B. subtilis KT004404 (Rehman et al., 2017) and Bacillus sp. CL33A (Ignatova et al., 1999). After 24 h of submerged fermentation of the strain in a bioreactor on minimal feather medium (0.3 g/Ll NaH2PO4, 0.35 g/l L Na2HPO4, 7.5 g/l L feather powder, pH 7.0) for 24 h, the strain culture supernatant demonstrated keratinase, protease, collagenase, milk-clotting, amylase, esterase, отформатировано: Шрифт: курсив отформатировано: Шрифт: курсив отформатировано: Шрифт: курсив phosphatase, and phytase activities. The specific activities and their respective substrates are detailed in Table 1 presents data on these activities with an indication of the substrate. For comparisoncomparative context, the keratinase activity of B. licheniformis ALW1 is 72.2 U/mL (Abdel-Fattah et al., 2018);, the protease activity of B. cereus FT and Bacillus sp. DPUA 1728 is 187 and 86.27 U/mL, respectively (Lima et al., 2015;Asha and 2018),; and the amylase activity of B. licheniformis 104.K is 163 U/mL (Kholikov et al., 2025). The Hhigh levels of keratinase, protease, and amylase, in combination with collagenase, amylase, esterase, phosphatase, and phytase activities, indicate that the potential of using the B. paralicheniformis T7 strain has significant potential as a producer source of multi-enzyme preparations (Aktayeva and Khassenov, 2024a). This strain is capable of effectively hydrolyzing keratin-containing raw materials, particularly bird feathers, horns, hooves, wool, and cattle skin, with resulting in the release of peptones and free amino acids (Aktayeva and Khassenov, 2024b). Keratinase While keratinase and protease activity were have also been observed in B. paralicheniformis MKU3, but this strain in contrast todiffers from B. paralicheniformis T7, B. paralicheniformis MKU3 by the lacks of collagenase activity (Santha Kalaikumari et al., 2019). During a five5-days of cultivation period on Difco sporulation mediamedium, Arrett-Kirschbaum agar, and or Modified modified Nutrient nutrient agar media, the strain produced endospore formations was observed, which showed resistance to capable of withstanding a temperature of 121 °C and a pressure of 1.1 bar for 20 minutes. Additionally, Tthis strain was sensitive to the following antibiotics: ampicillin, cefazolin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, kanamycin, nalidixic acid, penicillin, rifampicin, streptomycin, tetracycline, and tobramycin. . Zymographic analysis performed withusing protease inhibitors-: PMSF, EDTA, E64, and pepstatin A-and combined withcomplemented by proteomic studies, revealed that this strain secretes enzymes with a molecular weight masses of ranging from 20-to 60 kDa, . These enzymes predominantly belonging to the family of serine peptidases: S8 and S41 family families of serine peptidases (James, 1978), and metallopeptidases: the M14, M42, and M55 family families of metallopeptidases (Hazra et al., 2012),, with maximum peak activity observed at 60 °C and pH 9.0. The enzyme extract of the strain has amylase activity, which with reaches a maximum at pH 7.0 and at 85 °C. The strain's high production capacity and ability tobioreactor fermentation efficiency in a bioreactor, along with its protease and keratinase activities, make render it B. paralicheniformis T7 a promising candidate for industrial applications.The whole genome of B. paralicheniformis T7 was sequenced to acquire provide a broad and deepcomprehensive understanding of its genetic characteristics, as genomic sequences present an extensive viewpoint. Aseptic cultures of the strain were cultivated in 10 mL of nutritional broth (HiMedia, Mumbai, India) in a shaking incubator at 37 °C and 150 rpm for 18 h. After the cultivation process, the cells were gathered harvested by using centrifugal forcetion at a speed of 6000 × gtimes the acceleration due to gravity for a duration of 7 minutes at a temperature of 4 °C.De novo assembled assembly yielded a single circular contig with 45.93% G + C content was generated, measuring of 4,360,494 bp in length and exhibiting a high mean contig coverage of 360X (Supplementary File 1). Whole-genome assembly Aaccording tonnotation via the PATRIC DBdatabase, the whole genome assembly annotation identified a total of 4,652 proteincoding sequences in total, along with 82 tRNA molecules, 38 repetitive elements, and 24 rRNA molecules (Supplementary File 2). Among the identified proteins, The annotation also revealed 972 were classified as hypothetical, proteins and 3,680 proteins withwere assigned functional отформатировано: Шрифт: курсив отформатировано: Шрифт: курсив Отформатировано: Отступ: Первая строка: 1.27 см Добавлено примечание ([A5]): Please note that "as genomic sequences present an extensive viewpoint" was deleted as it is unnecessary. The intended meaning was also unclear.отформатировано: Шрифт: курсив отформатировано: Шрифт: курсив assignmentsroles,. Within the functional assignments,of which 1,099 proteins possessed were associated with Enzyme Commission (EC) numbers, 926 had with Gene Ontology (GO) annotationsterms, and 807 were mapped to KEGG pathways (Supplementary File 3). Comparative genomic analyses and phylogenetic tree reconstruction were performed for theusing Bacillus genus reference strains retrieveddownloaded from the NCBI database (Figure 2).The whole-genome assembly for B. paralicheniformis T7, which is available was deposited in NCBI GenBank, has data entry under accession number CP124861 with the relatedand BioProject number PRJNA967188. This assembly provided provides an extensive understanding overview of the genetic information contained in this strain together withand facilitates the identification of the specific genetic components responsible for the observed morphological characteristics. The analytical growth under genomic DNA Oxford Nanopore sequencing, de novo genome genome annotation, and genome genomic analysis were performed to process the The assembly was available in NCBI and is available support additional analyses and genome sequence of the B. paralicheniformis T7 strain was generated using Oxford Nanopore for the genome assembly of the genome. Nanopore sequencing the of without the for further DNA et al., et al., Furthermore, this method or for the this the of whole-genome sequencing et al., The of this sequencing method on the at which a DNA is through the and This has exhibited in whole-genome sequencing as in et al., 2024). the of and and were employed for genome and Hhigh sequencing coverage of sequencing was and the genome assembly yielded a single circular and Khassenov, B. Table Enzymatic activity of the supernatant from Bacillus paralicheniformis T7 after submerged All were performed and the average of the was reported as the with standard