Microbiological studies of hot springs in India: a review.

The earliest microbiological studies on hot springs in India date from 2003, a much later date compared to global attention in this striking field of study. As of today, 28 out of 400 geothermal springs have been explored following both culturable and non-culturable approaches. The temperatures and pH of the springs are 37-99 °C and 6.8-10, respectively. Several studies have been performed on the description of novel genera and species, characterization of different bio-resources, metagenomics of hot spring microbiome and whole genome analysis of few isolates. 17 strains representing novel species and many thermostable enzymes, including lipase, protease, chitinase, amylase, etc. with potential biotechnological applications have been reported by several authors. Influence of physico-chemical conditions, especially that of temperature, on shaping the hot spring microbiome has been established by metagenomic investigations. Bacteria are the predominant life forms in all the springs with an abundance of phyla Firmicutes, Proteobacteria, Actinobacteria, Thermi, Bacteroidetes, Deinococcus-Thermus and Chloroflexi. In this review, we have discussed the findings on all microbiological studies that have been carried out to date, on the 28 hot springs. Further, the possibilities of extrapolating these studies for practical applications and environmental impact assessment towards protection of natural ecosystem of hot springs have also been discussed.

Sulfitobacter faviae sp. nov., isolated from the coral Faviaveroni.

Three closely related, non-sporulating, aerobic, Gram-stain-negative, motile, rod-shaped isolates (S5-53T, S6-62 and S6-64) were obtained from mucus of corals Favia veroni from the Andaman Sea, India. Colonies grown on marine agar were small, circular and cream-coloured. Heterotrophic growth was observed at 10-40 °C and pH 6-10; optimum growth occurred at 25-30 °C and pH 7-8. 16S rRNA gene sequence analysis confirmed the isolates belonged to the genus Sulfitobacter and the three isolates shared more than 99 % pairwise sequence similarity. Strain S5-53T shared highest 16S rRNA gene sequence similarity of 98.43 % with Sulfitobacter dubius KMM 3554T. DNA-DNA relatedness among the three isolates was above 70 % whereas strain S5-53T showed less than 70 % relatedness with the type strains of closely related species. The DNA G+C content of strain S5-53T was 61 mol%. It contained phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and diphosphatidylglycerol as major polar lipids. Predominant fatty acids included C18 : 1ω7c, C18 : 1ω7c 11-methyl, C16 : 0 and C10 : 0 3-OH. Q10 was the major respiratory quinone. Based on this polyphasic analysis, the new isolates (S5-53T, S6-62 and S6-64) are considered to represent a novel species of the genus Sulfitobacter, for which the name Sulfitobacter faviae sp. nov. is proposed. The type strain is S5-53T(=JCM 31093T=LMG 29156T).

Comparative 16S rRNA signatures and multilocus sequence analysis for the genus Salinicola and description of Salinicola acroporae sp. nov., isolated from coral Acropora digitifera.

A novel Gram-negative, aerobic, motile marine bacterium, strain S4-41(T), was isolated from mucus of the coral Acropora digitifera from the Andaman Sea. Heterotrophic growth was observed in 0-25 % NaCl, at 15-45 °C and pH 4.5-9. In phylogenetic trees, strain S4-41(T) was grouped within the genus Salinicola but formed a separate branch distant from a cluster composed of Salinicola salarius M27(T) and Salinicola socius SMB35(T). DNA-DNA relatedness between strain S4-41(T) and these reference strains were well below 70 %. Q-9 was the sole respiratory quinone. The DNA G+C content was determined to be 63.6 mol%. Based on a polyphasic analysis, strain S4-41(T) is concluded to represent a novel species in the genus Salinicola for which the name Salinicola acroporae sp. nov. is proposed. The type strain is S4-41(T) (=JCM 30412(T) = LMG 28587(T)). Comparative 16S rRNA analysis of the genera Salinicola, Kushneria, Chromohalobacter and Cobetia revealed the presence of genus specific sequence signatures. Multilocus sequence analysis based on concatenated sequences of rRNAs (16S and 23S) and four protein coding housekeeping genes (atpA, gyrB, secA, rpoD) was found to be unnecessary for phylogenetic studies of the genus Salinicola.

Taxonomic study of the genus Tepidiphilus: transfer of Petrobacter succinatimandens to the genus Tepidiphilus as Tepidiphilus succinatimandens comb. nov., emended description of the genus Tepidiphilus and description of Tepidiphilus thermophilus sp. nov., isolated from a terrestrial hot spring.

Comparative phenotypic, chemotaxonomic and genetic analysis revealed significant similarities among strains of the genera Tepidiphilus and Petrobacter. Analysis of 16S rRNA gene sequences and DNA-DNA relatedness of the type strains Tepidiphilus margaritifer N2-214(T) and Petrobacter succinatimandens 4BON(T) showed sequence similarity of 98.9 % and less than 40 % relatedness, indicating that these strains represent different species of same genus. Both strains had phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and diphosphatidylglycerol as major polar lipids. Their fatty acid profiles were almost identical, with the predominant fatty acids C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c. In view of this, we propose to transfer the member of the genus Petrobacter to the genus Tepidiphilus as Tepidiphilus succinatimandens comb. nov. and to emend the description of the genus Tepidiphilus. Further, a novel bacterium, strain JHK30(T), was isolated from a terrestrial hot spring located at Jharkhand, India, and was identified following a polyphasic approach. Cells were non-sporulating, aerobic, Gram-stain-negative rods and motile by a single polar flagellum. Optimum temperature for growth was 50-55 °C at pH 6.5-7.0. 16S rRNA gene sequence analysis revealed 99.71 % similarity with P. succinatimandens 4BON(T) ( = DSM 15512(T)) and 98.71 % with T. margaritifer N2-214(T) ( = DSM 15129(T)). However, DNA-DNA relatedness of strain JHK30(T) with these two type strains was well below 70 %. The DNA G+C base composition was 66.1 mol%. Strain JHK30(T) represents a novel species of the genus Tepidiphilus for which the name Tepidiphilus thermophilus sp. nov. is proposed. The type strain is JHK30(T) ( = JCM 19170(T) = LMG 27587(T)= DSM 27220(T)).

Marinomonas fungiae sp. nov., isolated from the coral Fungia echinata from the Andaman Sea.

A novel aerobic marine bacterium, strain AN44(T), was isolated from the coral Fungia echinata sampled from the Andaman Sea, India. Cells were Gram-negative, motile and rod-shaped. Oxidase and catalase tests were positive. Heterotrophic growth was observed at pH 5.5-10 and at 16-42 °C, with optimum growth at pH 7-8 and 28 °C. Strain AN44(T) grew in the presence of 0.5-11% (w/v) NaCl; the optimal NaCl concentration for growth was 3-5%. The DNA G+C content was 47.8 mol%. Predominant cellular fatty acids of strain AN44(T) were C(18 : 1)ω7c, C(16 : 1)ω7c/C(16 : 1)ω6c, C(16 : 0), C(10 : 0) 3-OH, C(12 : 0), C(10 : 0), C(14 : 0) and C(18 : 0). The sole isoprenoid ubiquinone was Q-8. The polar lipids were an unidentified phospholipid, an unidentified aminophospholipid and two unidentified glycolipids. 16S rRNA gene sequence comparisons revealed that strain AN44(T) clustered within the radiation of the genus Marinomonas and showed similarity of 97.9% with Marinomonas ostreistagni UST010306-043(T), 97.8% with Marinomonas aquimarina 11SM4(T), 97.1% with Marinomonas brasilensis R-40503(T) and 97.0% with Marinomonas communis 8(T). However, DNA-DNA relatedness between strain AN44(T) and closely related type strains was well below 70%. On the basis of the data from the present polyphasic taxonomic study, strain AN44(T) is considered to represent a novel species of the genus Marinomonas, for which the name Marinomonas fungiae sp. nov. is proposed. The type strain is AN44(T) ( = JCM 18476(T) = LMG 27065(T)).

Photobacterium panuliri sp. nov., an alkalitolerant marine bacterium isolated from eggs of spiny lobster, Panulirus penicillatus from Andaman Sea.

A facultative anaerobe, alkalitolerant, gram-negative marine bacterium strain LBS5(T), was isolated from eggs carried on the pleopods of female spiny lobster (Panulirus penicillatus) in Andaman Sea from a depth of 3.5 m. Heterotrophic growth was observed at 15-38 °C and pH 5.5-11. Optimum growth occurred at 28 °C and pH 7.5. It can grow in the presence of 0.5-7 % NaCl (w/v), and the optimal NaCl required for growth was 2-4 %. 16S rRNA gene sequence analysis revealed the strain LBS5(T) belongs to the genus Photobacterium and showed 99.6 % similarity with P. aquae AE6(T), 98.2 % with P. aphoticum M46(T), 97 % with P. rosenbergii CC1(T), 96.9 % with P. lutimaris DF-42(T), and 96.6 % with P. halotolerans MACL01(T). The DNA-DNA similarities between strains LBS5(T) with other closely related strains were well below 70 %. The DNA G + C content was 50.52 (±0.9) mol%. The major fatty acids were C16:1w7c/w6c, C18:1w6c/w7c, C16:0, C15:0 iso, C16:0 10-methyl/17:1 iso w9c, C17:0 iso. Polar lipids included a phosphatidylglycerol, a diphosphatidylglycerol, a phosphatidylethanolamine, and one unidentified lipid. Based on the polyphasic evidences, strain LBS5(T) represents a novel species of the genus Photobacterium for which Photobacterium panuliri sp. nov. is proposed. The type strain is LBS5(T) (=DSM 27646(T) = LMG 27617(T) = JCM 19199(T)).

Artificial intelligence in parasitic disease control: A paradigm shift in health care.

Parasitic diseases, including malaria, leishmaniasis, and trypanosomiasis, continue to plague populations worldwide, particularly in resource-limited settings and disproportionately affecting vulnerable populations. It has limited the use of conventional health-care delivery and disease control approaches and necessitated exploring innovative strategies. In this direction, artificial intelligence (AI) has emerged as a transformative tool with immense promise in parasitic disease control, offering the potential for enhanced diagnostics, precision drug discovery, predictive modeling, and personalized treatment. Predictive AI algorithms have assisted in understanding parasite transmission patterns and outbreaks by analyzing vast amounts of epidemiological data, environmental factors, and population demographics. This has strengthened public health interventions, resource allocation, and outbreak preparedness strategies, enabling proactive measures to mitigate disease spread. In diagnostics, AI-enabled accurate and rapid identification of parasites by analyzing microscopic images. This capability is particularly valuable in remote regions with limited access to diagnostic facilities. AI-driven computational methods have also assisted in drug discovery for parasitic diseases by identifying novel drug targets and predicting the efficacy and safety of potential drug candidates. This approach has streamlined drug development, leading to more effective and targeted therapies. This article reviews these current developments and their transformative impacts on the health-care sector. It also assessed the hurdles that require attention before these transformations can be realized in real-life scenarios.

Deep tech innovation for parasite diagnosis: New dimensions and opportunities.

By converging advanced science, engineering, and design, deep techs are bringing a great wave of future innovations by mastering challenges and problem complexity across sectors and the field of parasitology is no exception. Remarkable research and advancements can be seen in the field of parasite detection and diagnosis through smartphone applications. Supervised and unsupervised data deep learnings are heavily exploited for the development of automated neural network models for the prediction of parasites, eggs, etc., From microscopic smears and/or sample images with more than 99% accuracy. It is expected that several models will emerge in the future wherein greater attention is being paid to improving the model's accuracy. Invariably, it will increase the chances of adoption across the commercial sectors dealing in health and related applications. However, parasitic life cycle complexity, host range, morphological forms, etc., need to be considered further while developing such models to make the deep tech innovations perfect for bedside and field applications. In this review, the recent development of deep tech innovations focusing on human parasites has been discussed focusing on the present and future dimensions, opportunities, and applications.

Enabling Trade in Gene-Edited Produce in Asia and Australasia: The Developing Regulatory Landscape and Future Perspectives.

Genome- or gene-editing (abbreviated here as 'GEd') presents great opportunities for crop improvement. This is especially so for the countries in the Asia-Pacific region, which is home to more than half of the world's growing population. A brief description of the science of gene-editing is provided with examples of GEd products. For the benefits of GEd technologies to be realized, international policy and regulatory environments must be clarified, otherwise non-tariff trade barriers will result. The status of regulations that relate to GEd crop products in Asian countries and Australasia are described, together with relevant definitions and responsible regulatory bodies. The regulatory landscape is changing rapidly: in some countries, the regulations are clear, in others they are developing, and some countries have yet to develop appropriate policies. There is clearly a need for the harmonization or alignment of GEd regulations in the region: this will promote the path-to-market and enable the benefits of GEd technologies to reach the end-users.

Molecular diagnosis of infectious parasites in the post-COVID-19 era.

The endemicity of several parasitic diseases across the globe and recent evidence of distress among COVID-19 patients with preexisting parasitic infections requires strengthening One Health framework and advanced strategies for parasitic detection. Owing to the greater sensitivity and accuracy, molecular technologies such as conventional polymerase chain reaction (PCR), reverse transcription (RT)-PCR, nested PCR, loop-mediated isothermal amplification (LAMP), and xMAP technology have been extensively studied for parasitic diagnosis. Varieties of genes have been targeted for primer development where 18S rRNA, internal transcribed spacer regions, and mitochondrial DNAs coding for cytochrome, and other enzymes have been widely used. More recent, low-cost sequencing and advances in big data management have resulted in a slow but steady rise of next-generation sequencing-based approaches for parasite diagnosis. However, except for few parasites of global concerns such as Plasmodium and Entamoeba, most of the molecular tools and technologies are yet to witness bench to bedside and field translations. This review looks into some of the advancements in the molecular diagnosis of parasites that have potential relevance to clinical purposes and may pave the way toward disease management in an efficient and timely manner.

Characterization of multidrug resistance in Vibrio species isolated from marine invertebrates from Andaman Sea.

This study describes the abundance of multidrug-resistant Vibrios associated with marine invertebrate hosts from the Andaman Sea, India. Thirty-eight Vibrio strains were isolated from surface mucus layers of coral Porites, Goniastrea, Pocillopora, Fungia, and eggs of spiny lobster (Panulirus penicillatus). Phenotypically, the majority of strains exhibited growth at a wide range of temperatures, salt tolerance, and diverse nutritional requirements. All the strains had more than 97% 16S rRNA sequence similarity with type species of the genus Vibrio where Vibrio fortis, and Vibrio alginolyticus were predominant. Multilocus Sequence Analysis (MLSA) using eight housekeeping genes namely ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA distributed the strains into 6 reported clades i.e., Harveyi, Ponticus, Nereis, Orientalis, Splendidus, and Mediterranei where nearly half of the total strains represented the clade Harveyi, followed by the clade Splendidus. Likewise, the PFGE profile indicated genomic heterogeneity among the strains resulting in their distribution in five major clusters. Resistance to different antimicrobials was tested following the disc diffusion method where all strains were found susceptible to chloramphenicol (30 µg) and resistant to streptomycin (10 µg), vancomycin (30 µg), sulfamethoxazole-trimethoprim (25 µg). Moreover, the resistant phenotype to other antimicrobials confirmed the abundance of multidrug resistance strains in this marine environment.

Cloning, Overexpression, and Characterization of Halostable, Solvent-Tolerant Novel ß-Endoglucanase from a Marine Bacterium Photobacterium panuliri LBS5(T) (DSM 27646(T)).

A 1329 nucleotide long endoglucanase gene was amplified from marine bacterium Photobacterium panuliri strain LBS5(T).The enzyme sequence was novel as protein-based similarity search revealed that it shared maximum similarity of 99% with hypothetical protein of P. aquae and 40% with endoglucanase of P. marinum AK15. The gene was cloned, overexpressed in Escherichia coli BL21 (DE3), and purified up to homogeneity using Ni-NTA affinity chromatography. The purified enzyme, designated as Cel8, was monomeric and has a molecular mass of 53 kDa. The enzyme was halostable and exhibited optimal carboxymethyl cellulase (CMCase) activity and stability at 2 M NaCl. Optimal activity was obtained at 40 °C and at pH 4. The enzyme exhibited remarkable stability in different organic solvents (50%, v/v), and activity increased nearly 1.5-fold in presence of butanol, isopropanol, petroleum ether, benzene, acetone, and n-hexane. It was active in Ca(2+), Ba(2+), and Ni(2+) and inhibited by Co(2+), Cd(2+), Zn(2+), Cu(2+), and Hg(2+). Under normal physiological conditions, the enzyme has 25% helix, 30% sheets, and 56% irregularities, whereas salt leads to helix to sheet transition in enzyme. Three-dimensional reconstruction analysis revealed that the enzyme has (α/ß)8 structure and a TIM barrel fold-like structure at the central groove of enzyme. This is the first evidenced report on halostable, organic solvent tolerant cellulase in the marine bacterial genus Photobacterium.

Draft Genome Sequence of Anoxybacillus suryakundensis Strain JS1T (DSM 27374T) Isolated from a Hot Spring in Jharkhand, India.

Anoxybacillus suryakundensis strain JS1(T), a facultative anaerobic, moderately thermophilic, alkalitolerant bacterium, was isolated from a hot spring. The estimated genome is 2.6 Mb and encodes 2,668 proteins.

Draft Genome Sequence of Idiomarina woesei Strain W11T (DSM 27808T) Isolated from the Andaman Sea.

Idiomarina woesei strain W11(T) was isolated from the Andaman Sea. Heterotrophic growth was observed at 30 to 37°C and pH 7 to 9. It grows in the presence of 0.5 to 12% (wt/vol) NaCl, and optimum growth occurred at 5 to 6% NaCl. The estimated genome is 2.4 Mb and encodes 2,305 proteins.

Draft Genome Sequence of Tepidiphilus thermophilus Strain JHK30T (JCM 19170T) Isolated from a Terrestrial Hot Spring in India.

Tepidiphilus thermophilus strain JHK30(T) was isolated from a hot spring at Surajkund, Jharkhand, India. It is a Gram-negative rod, nonsporulating, aerobic, and motile. The estimated genome is 2.3 Mb, with 2,186 protein-coding sequences.

Vibrio panuliri sp. nov., a marine bacterium isolated from spiny lobster, Panulirus penicillatus and transfer of Vibrio ponticus from Scophthalmi clade to the newly proposed Ponticus clade.

A novel marine bacterium, strain LBS2(T) was isolated from eggs carried on pleopods of the spiny lobster collected from Andaman Sea. Heterotrophic growth occurred at 1-7% NaCl. 16S rRNA gene sequence similarity revealed the strain LBS2(T) belonged to the genus Vibrio and showed above 97% similarity with eight type strains of the genus Vibrio. Multilocus analysis based on ftsZ, gapA, gyrB, mreB, pyrH recA, rpoA, and topA revealed LBS2(T) formed a separate cluster with Vibrio ponticus DSM 16217(T) with 89.8% multilocus gene sequence similarity. However, strain LBS2(T) is distantly related with other members of the Scophthalmi clade in terms of 16S rRNA signatures, phenotypic variations and multilocus gene sequence similarity, for which we propose LBS2(T) belongs to a new clade i.e. Ponticus clade with V. ponticus DSM 16217(T) as the representative type strain of the clade. DNA-DNA homologies between strain LBS2(T) and closely related strains were well below 70%. DNA G + C content was 45.3 mol%. On the basis of our polyphasic study, strain LBS2(T) represents a novel species of the genus Vibrio, for which the name Vibrio panuliri sp. nov. is proposed. The type strain is LBS2(T) (= JCM 19500(T) = DSM 27724(T) = LMG 27902(T)).

Comparative analysis of 16S rRNA signature sequences of the genus Idiomarina and Idiomarina woesei sp. nov., a novel marine bacterium isolated from the Andaman Sea.

A Gram-negative, short rod, aerobic bacterium, designated W11(T), was isolated from seawater. Heterotrophic growth was observed at 10-45 °C and pH 6-10. Optimal growth was observed at 30-37 °C and pH 7-9. It can grow in the presence of 0.5-12% NaCl (w/v), and the optimal NaCl required for growth was 5-6%. 16S rRNA gene sequence similarity revealed that strain W11(T) clustered within the radiation of the genus Idiomarina and showed 99.24% similarity with Idiomarina donghaiensis JCM 15533(T), 97.64% with Idiomarina marina JCM 15083(T), 97.37% with Idiomarina tainanensis JCM 15084(T) and 97.16% with Idiomarina maritima JCM 15534(T). DNA-DNA similarities between strains W11(T) with other closely related strains were below 70%. Polar lipids included a phosphatidylgylycerol, a diphosphatidylglycerol, a phosphatidylethanolamine, an unidentified phosopholipid, two unidentified aminolipids and two unidentified lipids. DNA G + C content was 41.2 ± 0.1 mol%. Major fatty acids were iso-C15:0, iso-C17:0, iso-C17:1ω9c, C16:0, iso-C11:0 3OH and C16:1ω7c/C16:1ω7c. The isoprenoid ubiquinone was Q8. On the basis of the present polyphasic taxonomic study, strain W11(T) is considered to represent a novel species of the genus Idiomarina, for which the name Idiomarina woesei sp. nov. is proposed. The type strain is W11(T) (= DSM 27808(T) = JCM 19499(T) = LMG 27903(T)).

Anoxybacillus suryakundensis sp. nov, a moderately thermophilic, alkalitolerant bacterium isolated from hot spring at Jharkhand, India.

Four closely related facultative anaerobe, moderately thermophilic, Gram positive rods (JS1(T), JS5, JS11, and JS15) were isolated from sediment samples from a hot spring at Suryakund, Jharkhand, India. Colonies were pale yellow, rough surface with uneven edges on TSA after 72 h incubation. Heterotrophic growth was observed at 40-60°C and pH 5.5-11.5; optimum growth occurred at 55°C and pH 7.5. 16S rRNA gene sequence analysis revealed the strains belong to genus Anoxybacillus. DNA-DNA homology values among strains were above 70% and showed distinct ERIC and REP PCR profile. On the basis of morphology and biochemical characteristics, strain JS1(T) was studied further. Strain JS1(T) showed 99.30% sequence similarity with A. flavithermus subsp. yunnanensis, 99.23% with A. mongoliensis, 99.16% with A. eryuanensis, 98.74% with A. flavithermus subsp. flavithermus, 98.54% with A. tengchongensis, 98.51% with A. pushchinoensis, 97.91% with A. thermarum, 97.82% with A. kaynarcensis, 97.77% with A. ayderensis and A. kamchatkensis, 97.63% with A. salavatliensis, 97.55% with A. kestanbolensis, 97.48% with A. contaminans, 97.27% with A. gonensis and 97.17% with A. voinovskiensis. In 16S rRNA secondary structure based phylogenetic comparison, strain JS1(T) was clustered with Anoxybacillus eryuanensis, A. mongoliensis, and A. flavithermus subsp. yunnanensis and showed 15 species specific base substitutions with maximum variability in helix 6. Moreover, DNA-DNA relatedness between JS1(T) and the closely related type strains were well below 70%. The DNA G+C content was 42.1 mol%. The major fatty acids were C(15:0 iso), C(16:0 iso) and C(17:0iso). The polar lipids were a phosphatidylgylycerol, a diphosphatidylglycerol, a phosphatidylethnolamine, a phosphatidylcholine, a phosphatidyl monomethylethanolamine and four unknown lipids. Based on polyphasic approach, strain JS1(T) represent a novel species of the genus Anoxybacillus for which Anoxybacillus suryakundensis sp. nov. is proposed. The type strain is JS1(T) (= DSM 27374(T) = LMG 27616(T) =JCM19211(T)).

Critical process parameters optimization for hyperthermostable ß amylase production by Bacillus subtilis DJ5 using response surface methodology / A otimização dos parâmetros do processo crítico para a produção hipertermoestável de ß amilase por Bacillus subtilis DJ5 utilizando a metodologia de superfície de resposta

The combined effects of significant physical and chemical factors affect hyperthermostable ß amylase production under submerged fermentation by Bacillus subtilis DJ5. The above was studied using the experimental design and response surface methodology. A 23 full-factorial central composite design was chosen to analyze interactions among three factors i.e. substrate concentration, medium pH and incubation temperature. The experimental data were fitted into a polynomial model for the yield of enzyme and an optimum level was arrived at with optimized conditions. Solving the coded values using Excel equation function indicated that maximum enzyme production is possible at a substrate concentration of 7.07 mg mL-1, pH 6.622 and temperature of 35.435°C. Such prediction was validated with practical experiments in which, at the prescribed condition maximum yield of 15.62 U mg-1, nearly 1.5 fold higher than non-optimized condition was observed.

Os efeitos combinados de fatores físicos e químicos significativos influenciam a produção hipertermoestável de amilase ß sob fermentação submersa por Bacillus subtilis DJ5. O esquema experimental e metodologia de superfície de resposta foram usados, com 23 planejamento fatorial composto, para analisar as interações entre os três fatores, ou seja, concentração de substrato, pH médio e temperatura de incubação. Os dados experimentais foram ajustados a um modelo polinomial para a produção de enzima e chegou-se a um nível ótimo através de condições otimizadas. A solução dos valores codificados por equação de Excel indicou que a produção máxima da enzima é possível a uma concentração de substrato de 7,07 mg mL-1, pH 6,622 e temperatura de 35,435°C. Tal previsão foi validada com experimentos práticos, onde na condição prescrita de rendimento máximo de 15,62 U mg-1, foram observados cerca de 1,5 vezes maior do que o estado não otimizado.