Maximum Protease Production Research Articles

Protease production and molecular characterization of a protease dipeptidyl-aminopeptidase gene from different strains of Sordaria fimicola.

The current research was designed to reach extracellular protease production potential in different strains of Sordaria fimicola which were previously obtained from Dr. Lamb (Imperial College, London) from North Facing Slope and South Facing Slope of Evolution Canyon. After initial and secondary screening, two hyper-producers strains S2 and N6 were selected for submerged fermentation and cultural conditions including temperature, pH, incubation period, inoculum size, substrate concentration, and different carbon and nitrogen sources were optimized for enzyme production. S2 strain showed maximum protease production of 3.291 U/mL after 14 days of incubation at 30 °C with 7 pH, 1% substrate concentration and 1 mL inoculum, While N6 strain showed maximum protease production of 1.929 U/mL under fermentation optimized conditions. Another aim of the present research was to underpin the biodiversity of genetics and post-translational modifications (PTMs) of protease DPAP (peptidyl-aminopeptidase) in Sordaria fimicola. Five polymorphic sites were observed in amino acid sequence of S. fimicola strains with reference to Neurospora crassa. PTMs prediction from bioinformatics tools predicted 38 phosphorylation sites on serine residues for protease peptidyl-aminopeptidase in S1 strain of S. fimicola while 45 phosphorylation sites on serine in N7 strain and 47 serine phosphorylation modifications were predicted in N. crassa. Current research gave an insight that change in genetic makeup effected PTMs which ultimately affected the production of protease enzyme in different strains of same organism (S. fimicola). The production and molecular data of the research revealed that environmental stress has strong effects on the specific genes through mutations which may cause genetic diversity. S. fimicola is non- pathogenic fungus and has a short life cycle. This fungus can be chosen to produce protease enzyme on a commercial scale.

Molecular Characterization and Optimization of Alkaline Protease Production by Bacillus cereus LS23B

The study aimed to determine the conditions leading to maximum protease production using submerged fermentation and detecting the presence of protease genes in the bacteria. This is necessary to meet the increasing demand for protease enzymes in the industrial market. The ability of the bacterial isolate to produce protease enzymes was evaluated through primary screening. After that, morphological characterization, biochemical tests, and 16S rRNA analysis were done to identify the bacterial strain. To confirm the presence of the gene-encoding enzyme, the protease (npr) gene primer was amplified using a polymerase chain reaction. The pH, incubation duration, temperature, carbon, and nitrogen sources were studied. Others include metal ions, substrate concentration, and agitation speed. The bacterial strain has 100% similarity to Bacillus cereus A9, while the protease encoding gene was confirmed with positive bands of 951bp. The enzyme was optimally produced at 40 oC with a pH of 9 after 72 h incubation. Starch, gelatin, 1 % substrate concentration, 1mM K+, and agitation speed of 160 rpm fully supported protease production. The presence of the npr gene in the isolate was confirmed. Also, the optimization study reveals that Bacillus cereus LS23B can be used in large-scale protease production, which may be used in different biotechnological applications.

Effect of Temperature and Fermentation Time on Protease Production Using Decapterus macarellus Fish Waste

The fish processing industries generate huge amounts of by-products which cause serious environmental and health problems. The environmental problems related to waste disposal can be reduced as the low cost of fish by-products contains nutrients that can enhance microbial growth and are useful for enzyme production. This research aims to investigate the effect of temperature and fermentation time on protease production from fish waste hydrolysate by using the Bacillus strain (Bacillus cereus). The pre-treatment and fish waste hydrolysate were carried out and continued with the production of protease. The effect of fermentation time was studied every 4 h for 72 h while the effect of temperature was investigated at temperatures ranging from 30 °C to 60 °C. The results showed the maximum protease production of 45.63 U/mL at 48 h of fermentation time and 44.908 ± 6.14 U/mL at a temperature of 50 °C. The current study provides insight into the effects of cultivation conditions on protease production from local fish waste industries for further optimization study to enhance protease production.

Isolation, identification, and optimization of the fibrinolytic protease-producing strains

Cardiovascular complications due to thrombosis have become one of the main causes of death worldwide. The high cost and undesirable side effects of existing thrombolytic agents have led researchers to isolate potential strains that produce fibrinolytic enzymes for therapeutic applications. Fibrinolytic enzymes, especially of microbial origin, are recognized as potential therapeutic candidates for thrombosis. In this study, isolation, identification, and optimization of fibrinolytic protease enzyme-producing strains were performed using fermentative protein sources. Fibrinolytic protease-producing strains were selected by analyzing the isolated strains on skim milk agar medium. The selected strains were examined on blood agar and fibrin plate medium, and the ones showing high enzymatic activity were determined. The strain determined to have the highest activity was identified as Acinetobacter johnsonii TR01 by 16S rRNA analysis. The maximum fibrinolytic protease production of the strain occurred at 60°C and pH 7.0. Under different medium conditions used for enzyme production, fructose was found to be the best carbon source, while yeast extract and peptone were the best nitrogen sources. It was observed that CaCl2, KH2PO4, andMgSO4 components had a negative effect, while MnCl2and ZnC4H6O4 components had a positive effect on enzyme production. The medium composition for maximum enzyme activity (8.30IU/ml) determined by Response Surface Methodology was 14.22g/L fructose, 11.190g/L yeast extract, 14.22g/L peptone, 0.5g/L MnCl2, and 0.5g/L ZnC4H6O4.

Isolation, screening and characterization of gelatinase and protease producing microorganisms from organic kitchen waste

The present study was aimed at isolation, screening and identification of gelatinase and protease producing bacteria from the kitchen waste. The study was also focused on the potential of isolates to produce enzyme protease and gelatinase. The total of 157 bacterial isolates were obtained from the organic kitchen waste collected from various nearby sources. The primary screening was done to screen the potential of these isolates for their enzymatic potentials on selective media. The 57 promising bacterial isolates were found to have enzymatic potentials for both gelatinase and protease. The screened isolates were subjected to secondary screening, amongst which five isolates showed maximum production of both gelatinase and protease. These five isolates viz. KW91, KW104, KW121, KW128, KW98 were the most promising isolates showing high degree of milk and gelatin hydrolysis which may be potent in faster degradation of domestic kitchen waste.

Optimization of Protease Production Using Agro Wastes by Aspergillus spp. In A Solid-State Fermentation

gricultural wastes are highly nutritious and facilitate microbial growth and metabolism. Most agro-wastes are lignocellulosic in nature, composed of lignin, hemicellulose and cellulose. Agricultural residues can be used for the production of various value-added products, such as industrially important enzymes. Extracellular proteases have high commercial value and multiple applications in detergents, food, dairy, pharmaceutical, leather, diagnostics, waste management and several recovery industries. Aspergillus oryzae was isolated from rice farmland, Rigasa, Kaduna. It was screened for protease production using skimmed milk agar assay with a wider clear zone of casein hydrolysis (halo zone). The main objectives of this study were to isolate, characterized, and optimize the production conditions by Aspergillus oryzae under solid state fermentation (SSF) using locally available agricultural wastes used as substrate. The results showed that the optimum conditions for maximum protease production are 84 hours of incubation; pH 6.0; temperature of 35ᵒC; 3% inoculum size; peptone as Nitrogen source and rice husk as the best fermentation substrate. The highest protease activity of rice husk was 1350 Ug−1 of dry solids compared with an activity of corn cob 1005 Ug−1 respectively. Protease was produced from Aspergillus oryzae with broad substrate specificity, high pH and temperature stability.

Development of a microbial protease for composting swine carcasses, optimization of its production and elucidation of its catalytic hydrolysis mechanism

BackgroundDead swine carcass composting is an excellent method for harmless treatment and resource utilization of swine carcass. However, poor biodegradation ability of traditional composting results in poor harmless treatment effect. Researches report that the biodegradation ability of composting can be improved by inoculation with enzyme-producing microorganisms or by inoculation with enzyme preparations. At present, the researches on improving the efficiency of dead swine carcass composting by inoculating enzyme-producing microorganisms have been reported. However, no work has been reported on the development of enzyme preparations for dead swine carcass composting.MethodologyThe protease-producing strain was isolated by casein medium, and was identified by 16 S rRNA gene sequencing. The optimal fermentation conditions for maximum protease production were gradually optimized by single factor test. The extracellular protease was purified by ammonium sulfate precipitation and Sephadex G-75 gel exclusion chromatography. The potential for composting applications of the purified protease was evaluated by characterization of its biochemical properties. And based on amino acid sequence analysis, molecular docking and inhibition test, the catalytic hydrolysis mechanism of the purified protease was elucidated.ResultsIn this study, a microbial protease was developed for swine carcass composting. A protease-producing strain DB1 was isolated from swine carcass compositing and identified as Serratia marcescen. Optimum fermentation conditions for maximum protease production were 5 g/L glucose, 5 g/L urea, 1.5 mmol/L Mg2+, initial pH-value 8, inoculation amount 5%, incubation temperature 30 °C and 60 h of fermentation time. The specific activity of purified protease reached 1982.77 U/mg, and molecular weight of the purified protease was 110 kDa. Optimum pH and temperature of the purified protease were 8 and 50 °C, respectively, and it had good stability at high temperature and in alkaline environments. The purified protease was a Ser/Glu/Asp triad serine protease which catalyzed substrate hydrolysis by Glu, Arg, Ser, Asp and Tyr active residues.ConclusionsIn general, the microbial protease developed in this study was suitable for industrial production and has the potential to enhance composting at thermophilic stage. Moreover, the catalytic hydrolysis mechanism of the protease was further analyzed in this study.

Valorization of by-products from vegetable oil industries: Enzymes production by Yarrowia lipolytica through solid state fermentation

Vegetable oil extraction generates high amounts of by-products, which are designated as oil cakes. Since the current strategies employed for oil cakes' reuse are linked with some drawbacks, identification of alternative approaches to decrease the environmental impact and promote a circular economy is of vital importance. In general, these materials are characterized by high fiber content, making them suitable to be employed in solid state fermentation (SSF). Filamentous fungi have been the microorganisms mostly applied in SSF and yeasts were applied in less extent. In the present work, three by-products from the extraction of olive, sunflower, and rapeseed oils were used as solid substrates in SSF for lipase and protease production by Yarrowia lipolytica W29. Oil cakes mixtures composition was optimized for the production of each enzyme using a simplex-centroid design of experiments. A 50% (w/w) mixture of olive cake (OC) and sunflower cake (SC) led to the highest lipase production, while a combination of the three oil cakes was most suitable for maximum protease production. Both enzymes were produced at maximum levels in a short period of 48 h. This work demonstrated that enzyme production by Y. lipolytica W29 in SSF can be modulated by the different combinations of oil cakes in the substrate mixture. Additionally, the potential of using by-products from vegetable oil industries in SSF processes was also demonstrated, showing alternative strategies for their valorization.

Screening and Characteristics of Marine Bacillus velezensis Z-1 Protease and Its Application of Enzymatic Hydrolysis of Mussels to Prepare Antioxidant Active Substances.

Bacillus velezensis is a type of microorganism that is beneficial to humans and animals. In this work, a protease-producing B. velezensis strain Z-1 was screened from sludge in the sea area near Qingdao (deposit number CGMCC No. 25059). The response surface methodology was used to analyze protease production, and the optimal temperature was 37.09 °C and pH 7.73 with the addition of 0.42% NaCl, resulting in maximum protease production of 17.64 U/mL. The optimum reaction temperature and pH of the protease of strain Z-1 were 60 °C and 9.0, respectively. The protease had good temperature and pH stability, and good stability in solvents such as methanol, ethanol and Tween 80. Ammonium, NH4+,and Mn2+ significantly promoted enzyme activity, while Zn2+ significantly inhibited the enzyme activity. The protease produced by strain Z-1 was used for the enzymolysis of mussel meat. The mussel hydrolysate exhibited good antioxidant function, with a DPPH free radical removal rate of 75.3%, a hydroxyl free radical removal rate of 75.9%, and a superoxide anion removal rate of 84.4%. This study provides a reference for the application of B. velez protease and the diverse processing applications of mussel meat.

Biotechnologically Important Enzyme Producing Indigenous Bacteria Isolated from Fruit and Vegetable Wastes Samples Collected from different Local Markets

Enzyme from bacterial sources is much stable and obtained cheaply. Amylases and proteases are among the most important enzymes. During this study, indigenous amylase and protease producing bacteria were isolated from common decayed fruits and vegetable wastes viz. Papaya, Brinjal, Cucumber, Potato and Snake Gourd. Bacterial load ranged in between 0.67 × 109 and 9.06 × 109 cfu/g on NA and 1.50 × 109 and 7.00 × 109 cfu/g on PYG agar medium. Maximum mean bacterial load on both NA (6.19 ± 2.60 × 109 cfu/g) and PYG agar (4.54 ± 1.03 × 109 cfu/g) were observed in decayed papaya. A total of 113 bacterial isolates were primarily isolated. Considering better amylase and protease activity 16 isolates were selected for detailed study. The starch hydrolysis ratio (SHR) of the isolates ranged in between 1.25 ± 0.37 and 2.47 ± 0.23 while casein hydrolysis ratio (CHR) ranged in between 2.35 ± 0.12 and 6.44 ± 1.16. The highest SHR was 2.47 ± 0.23 found in Bacillus sp. of snake gourd and the highest CHR was 6.44 ± 1.16 found in Bacillus subtilis also from snake gourd. Out of the 16 isolates 13 were Gram positive and 3 were Gram negative. Gram positive isolates were identified as Bacillus acidocaldarius (4), B. firmus (2), B. lentus (4), B. subtilis (2) and B. alcalophilus (1). Gram negative isolates were identified as Edwardsiella hoshinae, Proteus myxofaciens and P. mirabilis. Six isolates having higher SHR and CHR were authenticated through molecular identification and were identified as Chryseobacterium sp. S29.2, Bacillus sp. X8, Bacillus sp. strain GA1B, Bacillus sp.TdEND26, Bacillus subtilis strain BPA28 and Bacillus subtilis BAB-881. The maximum amylase (61.33 ± 2.14 U/ml) and protease (56.91 ± 0.23 U/ml) production were observed in Bacillus sp. TdEND26 rd. In case of co-production, the highest amount of amylase (54.13 ± 1.23 U/ml) protease (81.80 ± 4.54 U/ml) production was observed in Bacillus sp. TdEND26 at 24 hrs. Plant Tissue Cult. & Biotech. 32(1): 31-41, 2022 (June)

Dairy wastewater treatment using immobilized bacteria on calcium alginate in a microbial electrochemical system

The present study was conducted to assess the effect of immobilized Pseudomonas aeruginosa at calcium alginate beads for improving protease and lipase production as the main biocatalysts for dairy wastewater treatment in a microbial electrochemical system. A single-chamber cylindrical bioreactor with adequate volume of 3 L equipped with circular steel mesh and carbon cloth as the cathode and anode electrodes was utilized for the experiments. The main operating variables, including percent of alginate beads, pH, concentration, current density, and time were studied. The obtained results showed that the maximum production of protease and lipase in pH = 6.0 and induced current of 8.0 mA were 193.81 and 47.89 IU/L, respectively. The best precentage of calcium alginate in the bioreactor were obtained 40%. Moreover, the enzyme production time compared to the non-alginate reactor decreased from 12 to 4 h. According to the obtained results, the immobilization of Pseudomonas aeruginosa on calcium alginate along with the electrical stimulation of the bacteria increases the enzyme activities and thus reduces the time of treatment of dairy wastewater in the microbial electrochemical system.

Efficacy of Aspergillus sp in the Production of Protease Enzyme with Different Substrates

In the present study, the soil samples were collected from marine environment of Arichalmunai, Dhanushkodi, Ramnad District, Tamilnadu ,India. Fungal species were isolated by plating method, in 50% sea water containing potato dextrose agar medium .Totally 16 fungal species were isolated and identified from the soil sample. The production of protease from Aspergillus niger , A.flavus and A. terreus by liquid state fermentation. The production of protease enzyme was optimized by using fermentation medium containing different substrates. The maximum protease production was observed on wheat bran, containing medium. The protease production was maximum in temperature 35ºC were recorded. Wheat bran produced the maximum level protease. The optimization work also carried out. This study revealed that coastal environment provides impressive density of fungi in the East Coast of India and are unexplored for microbial resources can be useful in industry.

Optimization and Functional Properties of a Protease from a Fish Gut Isolate Pseudomonas sp. PD14 Grown on Fish Processing Wastes Substrate

Extracellular protease production from Pseudomonas sp. PD14 isolated from the gut of rabbitfish, Siganus guttatus, was investigated using fish processing wastes as nitrogen source. Three types of fish processing by-products – namely, heads, bones, and viscera – from yellowstripe scad (Selaroides sp.) and Japanese halfbeak (Hyporhampus sajori), as well as trimmings and viscera from squid (Loligo sp.) were used. Among the fish waste preparations tested, Japanese halfbeak viscera (HV) flour (1.0%) supported the maximum protease production of 365.0 U/mg protein. Effects of media components on protease production in the optimized concentration of HV flour revealed that the protease enzyme can be optimally produced using maltose (0.50%) as a carbon source, at pH 8.0, and incubated for 48 h at 40 °C. Partial characterization of the crude enzyme supernatant showed that the optimum pH and temperature were 8.0 and 50 °C, respectively. Furthermore, the crude protease exhibited thermostability retaining 50% activity at 60 °C and fairly stable up to pH 11.0. Considering the high costs of industrial enzyme production, the use of fish processing wastes for protease production offers a promising future for enzyme biotechnology.

Bio-production of alkaline protease by Trichoderma longibrachiatum and Penicillium rubidurum using different agro-industrial products

Alkaline protease being active in neutral to alkaline pH has huge demands in food, detergent, leather and pharmaceutical industries. Its production from agro-industrial wastes not only lowers the production costs but also reduces the environmental problems. Hence, the present study aimed to search for new potential microbes, which can produce alkaline protease enzyme, to meet the industrial demands. In this study, 13 fungal spp. were isolated on potato dextrose agar medium (PDA) from mangrove soil through serial dilution, and then were streaked on the skim milk agar medium for qualitative screening of protease production. Out of 13 fungal spp.; only 7 spp. were able to produce proteolytic zones through the proteolytic assay. The Relative enzymatic index (REI) value (Zone diameter/Colony diameter) of all the fungal isolates that produced proteolytic zones on skim milk agar medium was evaluated. Only 2 fungal isolates which showed maximum REI value were selected, and then identified morphologically and molecularly as Trichoderma longibrachiatum (Accession no. MF144551) and by Penicillium rubidurum (Accession no. MF144561). Submerged fermentation was carried out using different agro industrial substrates to quantify for protease production, where the supernatants obtained were used for alkaline protease estimation. Among the different tested substrates, soybean powder and wheat bran were the most suitable substrates for maximum protease production by T. longibrachiatum (233.78±7.12 U/ mg) and P. rubidurum (228.61±11.13 U/ mg), respectively. The partial purified enzyme from these fungi showed maximum proteolytic potentials at pH 8.0 (P. rubidurum) and pH 9.0 (T. longibrachiatum), with optima temperature of 40 °C. Among the tested heavy metals, only Mn2+ expressed marginal enhancement of the protease enzyme activity.

Substrate preferences, phylogenetic and biochemical properties of proteolytic bacteria present in the digestive tract of Nile tilapia (Oreochromis niloticus).

Vertebrate intestine appears to be an excellent source of proteolytic bacteria for industrial and probiotic use. We therefore aimed at obtaining the gut-associated proteolytic species of Nile tilapia (Oreochromis niloticus). We have isolated twenty six bacterial strains from its intestinal tract, seven of which showed exoprotease activity with the formation of clear halos on skim milk. Their depolymerization ability was further assessed on three distinct proteins including casein, gelatin, and albumin. All the isolates could successfully hydrolyze the three substrates indicating relatively broad specificity of their secreted proteases. Molecular taxonomy and phylogeny of the proteolytic isolates were determined based on their 16S rRNA gene barcoding, which suggested that the seven strains belong to three phyla viz. Firmicutes, Proteobacteria, and Actinobacteria, distributed across the genera Priestia, Citrobacter, Pseudomonas, Stenotrophomonas, Burkholderia, Providencia, and Micrococcus. The isolates were further characterized by a comprehensive study of their morphological, cultural, cellular and biochemical properties which were consistent with the phylogenetic annotations. To reveal their proteolytic capacity alongside substrate preferences, enzyme-production was determined by the diffusion assay. The Pseudomonas, Stenotrophomonas and Micrococcus isolates appeared to be most promising with maximum protease production on casein, gelatin, and albumin media respectively. Our findings present valuable insights into the phylogenetic and biochemical properties of gut-associated proteolytic strains of Nile tilapia.

Purification and characterization of an extracellular alkaline cold-adapted serine metalo-protease from the cold tolerant bacterium, Stenotrophomonas sp. BTR88

Proteases are the most economical enzymes in biotechnology and industry. Nowadays, a lot of attention is being paid to extremophiles microorganisms owing to the diversity of their enzymes. One hundred and Six proteolytic bacteria were isolated from Binaloud Mountain; one of them (strain BTR88) was selected as the best producer of extracellular protease and was used for further studies. This bacterium belongs to Stenotrophomonas sp., which were identified by the 16S rDNA sequence. Maximal protease production was detected at the beginning of exponential growth phase in the presence of starch and skim milk at 20°C and pH 9. This protease was purified to electrophoretic homogeneity with a fold: 27.5, yield: 33% and specific enzyme: 12.6 U/mg. SDS-PAGE and zymography analyses revealed a protein band of 22 kDa. The maximum activity was at pH 9 and in the range of 20-30°C; while, the enzyme exhibited a broad range of activity from 20-80°C as well as the pH of 5-10. Enzyme inhibition in the presence of phenylmethane sulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA) showed that the purified enzyme belongs to serine metallo-enzymes. Kinetic parameters of km, Vmax and kcat for the cold tolerant enzyme were determined to be 7.2 mg/mL, 1.45 mM/min and 33.2 sec−1, respectively. The characteristics of activity in cold and alkaline conditions and the broad range of pH and temperature suggest that serine metalloprotease has potential use in the detergent industry.

Two steps purification, biochemical characterization, thermodynamics and structure elucidation of thermostable alkaline serine protease from Nocardiopsis alba strain OM-5

The Nocardiopsis alba strain OM-5 showed maximum protease production in submerged culture. The OM-5 protease was purified by hydrophobic interaction chromatography. The purified protease of 68 kDa showed maximum activity (3312 ± 1.64 U/mL) at 70 °C and was quite stable at 80 °C up to 4 M NaCl (w/v) at pH 9. The purified protease showed significant activity and stability in different cations, denaturing agents, metal ions, and osmolytes. The thermodynamic parameters including deactivation rate constant (Kd) and half lives (t1/2) at 50–80 °C were in the range of 2.50 × 10−3 to 5.50 × 10−3 and 277.25–111.25 min respectively at 0–4 M NaCl. The structural stability of the OM-5 protease under various harsh conditions was elucidated by circular dichroism (CD) spectroscopy followed by K2D3 analysis revealed that the native structure of OM-5 protease was stable even in sodium dodecyl sulfate and Tween 20 indicated by increased α-helices content assisted with decreased β-sheets content.

Hydrolytic enzymes and biodeterioration of groundnut seeds

Some common seed borne fungal isolates of Groundnut variety TAG-24 and SB-XI were screened for their ability to produce the hydrolytic enzymes. For this the test seed borne fungal isolates of Groundnut were grown on substrate and nonsubstrate glucose nitrate media for seven days at room temperature. After seven days of incubation the culture filtrates were collected separately and considered them as the crude enzyme preparation. The crude enzyme preparations were assayed by different methods under different conditions. Both varieties of Groundnut Aspergillus terreus and Sclerotium rolfsii were found to be highly amlolytic. All the test isolates of the Groundnut variety TAG-24 and SB-XI produced protease in both the media. Maximum protease production was found by Aspergillus flavus followed by Fusarium oxysporum. All the test isolates produced cellulase in both the media. Maximum cellulase production was found in Sclerotium rolfsii followed by Aspergillus niger and Aspergillus terreus. Among the seven test isolates Aspergillus flavus, Aspergillus terreus, Macrophomina phaseolina could not produce pectinase in the absence of substrate but the production was observed in the presence of substrate

Protease production by Aspergillus flavus under solid state fermentation using cotton seeds and sugarcane bagasse

The production of protease by Aspergillus flavus under optimized conditions has been studied under solid state fermentation. Cotton seeds and sugarcane bagasse is used as substrates. The best substrate for maximum protease production was found to be sugarcane bagasse and a high protease activity was observed on the 144th hr of growth. A pH value of 4.0 with temperature 40 0C, inoculums size of 3% of solid substrate and a moisture content of 60% were found to be optimal for maximum protease production. The study proved that locally isolated fungal species is able to produce a very high level of protease under SSF using inexpensive agro-residual substrates, which can be useful in industries.

Effect of Cultural Conditions on Protease Production by a Thermophilic Geobacillus thermoglucosidasius SKF4 Isolated from Sungai Klah Hot Spring Park, Malaysia.

Major progress in the fields of agriculture, industry, and biotechnology over the years has influenced the quest for a potent microorganism with favorable properties to be used in scientific research and industry. This study intended to isolate a new thermophilic-protease-producing bacterium and evaluate its growth and protease production under cultural conditions. Protease producing bacteria were successfully isolated from Sungai Klah Hot Spring Park in Perak, Malaysia, and coded as SKF4; they were promising protease producers. Based on microscopic, morphological, and 16S rRNA gene analysis, isolate SKF4 was identified as Geobacillus thermoglucosidasius SKF4. The process of isolating SKF4 to grow and produce proteases under different cultural conditions, including temperature, pH, NaCl concentration, carbon and nitrogen sources, and incubation time, was explored. The optimum cultural conditions observed for growth and protease production were at 60 to 65 °C of temperature, pH 7 to 8, and under 1% NaCl concentration. Further, the use of casein and yeast extract as the nitrogen sources, and sucrose and fructose as the carbon sources enhanced the growth and protease production of isolate SKF4. Meanwhile, isolate SKF4 reached maximum growth and protease production at 24 h of incubation time. The results of this study revealed a new potent strain of thermophilic bacterium isolated from Sungai Klah Hot Spring Park in Perak, Malaysia for the first time. The high production of thermostable protease enzyme by G. thermoglucosidasius SKF4 highlighted the promising properties of this bacterium for industrial and biotechnological applications.