Secretory expression of amylosucrase in Bacillus licheniformis through twin-arginine translocation pathway.

Amylosucrase (EC 2.4.1.4) is a versatile enzyme with significant potential in biotechnology and food production. To facilitate its efficient preparation, a novel expression strategy was implemented in Bacillus licheniformis for the secretory expression of Neisseria polysaccharea amylosucrase (NpAS). The host strain B. licheniformis CBBD302 underwent genetic modification through the deletion of sacB, a gene responsible for encoding levansucrase that synthesizes extracellular levan from sucrose, resulting in a levan-deficient strain, B. licheniformis CBBD302B. Neisseria polysaccharea amylosucrase was successfully expressed in B. licheniformis CBBD302B using the highly efficient Sec-type signal peptide SamyL, but its extracellular translocation was unsuccessful. Consequently, the expression of NpAS via the twin-arginine translocation (TAT) pathway was investigated using the signal peptide SglmU. The study revealed that NpAS could be effectively translocated extracellularly through the TAT pathway, with the signal peptide SglmU facilitating the process. Remarkably, 62.81% of the total expressed activity was detected in the medium. This study marks the first successful secretory expression of NpAS in Bacillus species host cells, establishing a foundation for its future efficient production. ONE-SENTENCE SUMMARY: Amylosucrase was secreted in Bacillus licheniformis via the twin-arginine translocation pathway.

High-efficiency chromosomal integrative amplification strategy for overexpressing α-amylase in Bacillus licheniformis.

Bacillus licheniformis is a well-known platform strain for production of industrial enzymes. However, the development of genetically stable recombinant B. licheniformis for high-yield enzyme production is still laborious. Here, a pair of plasmids, pUB-MazF and pUB'-EX1, were firstly constructed. pUB-MazF is a thermosensitive, self-replicable plasmid. It was able to efficiently cure from the host cell through induced expression of an endoribonuclease MazF, which is lethal to the host cell. pUB'-EX1 is a nonreplicative and integrative plasmid. Its replication was dependent on the thermosensitive replicase produced by pUB-MazF. Transformation of pUB'-EX1 into the B. licheniformis BL-UBM harboring pUB-MazF resulted in both plasmids coexisting in the host cell. At an elevated temperature, and in the presence of isopropyl-1-thio-ß-d-galactopyranoside and kanamycin, curing of the pUB-MazF and multiple-copy integration of pUB'-EX1 occurred, simultaneously. Through this procedure, genetically stable recombinants integrated multiple copies of amyS, from Geobacillus stearothermophilus ATCC 31195 were facilely obtained. The genetic stability of the recombinants was verified by repeated subculturing and shaking flask fermentations. The production of α-amylase by recombinant BLiS-002, harboring five copies of amyS, in a 50-l bioreactor reached 50 753 U/ml after 72 hr fermentation. This strategy therefore has potential for production of other enzymes in B. licheniformis and for genetic modification of other Bacillus species.

Exploitation of ammonia-inducible promoters for enzyme overexpression in Bacillus licheniformis.

Ammonium hydroxide is conventionally used as an alkaline reagent and cost-effective nitrogen source in enzyme manufacturing processes. However, few ammonia-inducible enzyme expression systems have been described thus far. In this study, genomic-wide transcriptional changes in Bacillus licheniformis CBBD302 cultivated in media supplemented with ammonia were analyzed, resulting in identification of 1443 differently expressed genes, of which 859 genes were upregulated and 584 downregulated. Subsequently, the nucleotide sequences of ammonia-inducible promoters were analyzed and their functionally-mediated expression of amyL, encoding an α-amylase, was shown. TRNA_RS39005 (copA), TRNA_RS41250 (sacA), TRNA_RS23130 (pdpX), TRNA_RS42535 (ald), TRNA_RS31535 (plp), and TRNA_RS23240 (dfp) were selected out of the 859 upregulated genes and each showed higher transcription levels (FPKM values) in the presence of ammonia and glucose than that of the control. The promoters, PcopA from copA, PsacA from sacA, PpdpX from pdpX, Pald from ald, and Pplp from plp, except Pdfp from dfp, were able to mediate amyL expression and were significantly induced by ammonia. The highest enzyme expression level was mediated by Pplp and represented 23% more α-amylase activity after induction by ammonia in a 5-L fermenter. In conclusion, B. licheniformis possesses glucose-independent ammonia-inducible promoters, which can be used to mediate enzyme expression and therefore enhance the enzyme yield in fermentations conventionally fed with ammonia for pH adjustment and nitrogen supply.

Applications of weighted association networks applied to compositional data in biology.

Next-generation sequencing technologies have generated, and continue to produce, an increasingly large corpus of biological data. The data generated are inherently compositional as they convey only relative information dependent upon the capacity of the instrument, experimental design and technical bias. There is considerable information to be gained through network analysis by studying the interactions between components within a system. Network theory methods using compositional data are powerful approaches for quantifying relationships between biological components and their relevance to phenotype, environmental conditions or other external variables. However, many of the statistical assumptions used for network analysis are not designed for compositional data and can bias downstream results. In this mini-review, we illustrate the utility of network theory in biological systems and investigate modern techniques while introducing researchers to frameworks for implementation. We overview (1) compositional data analysis, (2) data transformations and (3) network theory along with insight on a battery of network types including static-, temporal-, sample-specific- and differential-networks. The intention of this mini-review is not to provide a comprehensive overview of network methods, rather to introduce microbiology researchers to (semi)-unsupervised data-driven approaches for inferring latent structures that may give insight into biological phenomena or abstract mechanics of complex systems.

Biotechnological potential of Beauveria bassiana as a source of novel biocatalysts and metabolites.

Beauveria bassiana though widely perceived as an entomopathogenic fungus has also been found in nature to be endophytic. As entomopathogens, the life cycle of different B. bassiana strains are organized and adapted as pathogens to their invertebrate hosts while as endophytes they maintain a symbiotic relationship with their plant hosts. To fulfill these aforementioned ecological roles, this fungus secretes an array of enzymes as well as secondary metabolites, which all have significant biological roles. Basically, chitinases, lipases and proteases are considered to be the most important of all the enzymes produced by B. bassiana. However, studies have also shown their ability to produce other vital enzymes which include amylase, asparaginase, cellulase, galactosidase etc. Previous reports on this filamentous fungus have laid more emphasis on its entomopathogenicity, its endophytism and its highly acclaimed application in the biological control of pests. This review, however, is the first to fully assess the enzyme-secreting potential of this entomopathogenic fungus and its use as a novel source of several industrial biocatalysts and other important biochemicals. This article highlights the inherent properties of the fungus to degrade various biopolymers as well as its relative safety for human use. Some of the important factors have raised the possibilities of exploitation for industrial production and as safe hosts for gene expression.

Recent advances in microbial glutaminase production and applications-a concise review.

This article focuses on significant advances in the production and applications of microbial glutaminases and provides insight into the structures of different glutaminases. Glutaminases catalyze the deamidation of glutamine to glutamic acid, and this unique ability forms the basis of their applications in various industries such as pharmaceutical and food organizations. Microbial glutaminases from bacteria, actinomycetes, yeast, and fungi are of greater significance than animal glutaminases because of their stability, affordability, and ease of production. Owing to these notable benefits, they are considered to possess considerable potential in anticancer and antiviral therapy, flavor enhancers in oriental foods, biosensors and in the production of a nutraceutical theanine. This review also aims to fully explore the potential of microbial glutaminases and to set the pace for future prospects.

Improved ethanol productivity from lignocellulosic hydrolysates by Escherichia coli with regulated glucose utilization.

BACKGROUND: Lignocellulosic ethanol could offer a sustainable source to meet the increasing worldwide demand for fuel. However, efficient and simultaneous metabolism of all types of sugars in lignocellulosic hydrolysates by ethanol-producing strains is still a challenge. RESULTS: An engineered strain Escherichia coli B0013-2021HPA with regulated glucose utilization, which could use all monosaccharides in lignocellulosic hydrolysates except glucose for cell growth and glucose for ethanol production, was constructed. In E. coli B0013-2021HPA, pta-ackA, ldhA and pflB were deleted to block the formation of acetate, lactate and formate and additional three mutations at glk, ptsG and manZ generated to block the glucose uptake and catabolism, followed by the replacement of the wild-type frdA locus with the ptsG expression cassette under the control of the temperature-inducible λ pR and pL promoters, and the final introduction of pEtac-PA carrying Zymomonas mobilis pdc and adhB for the ethanol pathway. B0013-2021HPA was able to utilize almost all xylose, galactose and arabinose but not glucose for cell propagation at 34 °C and converted all sugars to ethanol at 42 °C under oxygen-limited fermentation conditions. CONCLUSIONS: Engineered E. coli strain with regulated glucose utilization showed efficient metabolism of mixed sugars in lignocellulosic hydrolysates and thus higher productivity of ethanol production.

A novel strategy for production of ethanol and recovery of xylose from simulated corncob hydrolysate.

OBJECTIVES: To develop a xylose-nonutilizing Escherichia coli strain for ethanol production and xylose recovery. RESULTS: Xylose-nonutilizing E. coli CICIM B0013-2012 was successfully constructed from E. coli B0013-1030 (pta-ack, ldhA, pflB, xylH) by deletion of frdA, xylA and xylE. It exhibited robust growth on plates containing glucose, arabinose or galactose, but failed to grow on xylose. The ethanol synthesis pathway was then introduced into B0013-2012 to create an ethanologenic strain B0013-2012PA. In shaking flask fermentation, B0013-2012PA fermented glucose to ethanol with the yield of 48.4 g/100 g sugar while xylose remained in the broth. In a 7-l bioreactor, B0013-2012PA fermented glucose, galactose and arabinose in the simulated corncob hydrolysate to 53.4 g/l ethanol with the yield of 48.9 g/100 g sugars and left 69.6 g/l xylose in the broth, representing 98.6% of the total xylose in the simulated corncob hydrolysate. CONCLUSIONS: By using newly constructed strain B0013-2012PA, we successfully developed an efficient bioprocess for ethanol production and xylose recovery from the simulated corncob hydrolysate.

Identification and characterization of the steroid 15α-hydroxylase gene from Penicillium raistrickii.

Penicillium raistrickii ATCC 10490 is used for the commercial preparation of 15α-13-methy-estr-4-ene-3,17-dione, a key intermediate in the synthesis of gestodene, which is a major component of third-generation contraceptive pills. Although it was previously shown that a cytochrome P450 enzyme in P. raistrickii is involved in steroid 15α-hydroxylation, the gene encoding the steroid 15α-hydroxylase remained unknown. In this study, we report the cloning and characterization of the 15α-hydroxylase gene from P. raistrickii ATCC 10490 by combining transcriptomic profiling with functional heterologous expression in Saccharomyces cerevisiae. The full-length open reading frame (ORF) of the 15α-hydroxylase gene P450pra is 1563 bp and predicted to encode a cytochrome P450 protein of 520 amino acids. Targeted gene deletion revealed that P450pra is solely responsible for 15α-hydroxylation activity on 13-methy-estr-4-ene-3,17-dione in P. raistrickii ATCC 10490. The identification of the 15α-hydroxylase gene from P. raistrickii should help elucidate the molecular basis of regio- and stereo-specificity of steroid 15α-hydroxylation and aid in the engineering of more efficient industrial strains for useful steroid 15α-hydroxylation reactions.

Limitation of thiamine pyrophosphate supply to growing Escherichia coli switches metabolism to efficient D-lactate formation.

Efficient production of D-lactate by engineered Escherichia coli entails balancing cell growth and product synthesis. To develop a metabolic switch to implement a desirable transition from cell growth to product fermentation, a thiamine auxotroph B0013-080A was constructed in a highly efficient D-lactate producer E. coli strain B0013-070. This was achieved by inactivation of thiE, a gene encoding a thiamine phosphate synthase for biosynthesis of thiamine monophosphate. The resultant mutant B0013-080A failed to grow on the medium in the absence of thiamine yet growth was restored when exogenous thiamine was provided. A linear relationship between cell mass formation and amount of thiamine supplemented was mathematically determined in a shake flask experiment and confirmed in a 7-L bioreactor system. This calculation revealed that ∼ 95-96 thiamine molecules per cell were required to satisfy cell growth. This relationship was employed to develop a novel fermentation process for D-lactate production by using thiamine as a limiting condition. A D-lactate productivity of 4.11 g · L(-1) · h(-1) from glycerol under microaerobic condition and 3.66 g · L(-1) · h(-1) from glucose under anaerobic condition was achieved which is 19.1% and 10.2% higher respectively than the parental strain. These results revealed a convenient and reliable method to control cell growth and improve D-lactate fermentation. This control strategy could be applied to other biotechnological processes that require optimal allocation of carbon between cell growth and product formation.

Prevalence of leprosy-related disability in Bangladesh.

Disability due to leprosy often lasts lifelong, so estimates of the burden of leprosy in a community based on 'registered prevalence of leprosy cases' or on 'proportion with Grade 2 disability amongst new cases in past year' will seriously underestimate the number of disabled people in the community needing support or services. In a previously highly endemic are of Bangladesh, the accumulated prevalence of disability due to leprosy amongst adults was 45·35/100,000 population.

Production, characteristics and applications of phytase from a rhizosphere isolated Enterobacter sp. ACSS.

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40-80 °C) and pH (2.0-6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K m, V max, K cat, and K cat/K m of the pure phytase were 0.21 mM, 131.58 nmol mg(-1) s(-1), 1.64 × 10(3) s(-1), and 7.81 × 10(6) M(-1) s(-1), respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca(+2), Mg(+2) and Mn(+2), but inhibited by Zn(+2), Cu(+2), Fe(+2), Pb(+2), Ba(+2) and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.

Thermostable chitinase II from Thermomyces lanuginosus SSBP: Cloning, structure prediction and molecular dynamics simulations.

Thermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65kDa. Our study reports that chitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/ß domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350K.

Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications.

Chitinases are ubiquitous class of extracellular enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, chitinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, environmental and biotechnological applications due to their high stability for temperature and pH. Thermomyces lanuginosus produced a large number of chitinases, of which chitinase I and II are successfully cloned and purified recently. Molecular dynamic simulations revealed that the stability of these enzymes are maintained even at higher temperature. In this review article we have focused on chitinases from different sources, mainly fungal chitinase of T. lanuginosus and its industrial application.

Purification and Characterization of an Endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 Mutant.

An extracellular endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 mutant was purified to homogeneity by gel filtration chromatography and showed a specific activity of 119 U/mg. The optimum pH and temperature of the purified enzyme were found to be 6.0 and 50 °C, respectively. The enzyme was stable up to 60 °C, retaining 60% of residual activity for 30 min, but inactivated rapidly above 60 °C. The enzyme was found to be stable at pH=6-9 when it retained 100% of its residual activity. The Lineweaver-Burk plot showed that the apparent Km and vmax values of the inulinase when using inulin as a substrate were 1.15 mg/mL and 0.15 µM/min, respectively, whereas the kcat value was found to be 0.145 min-1. The calculated catalytic efficiency of the enzyme was found to be 0.126 (mg·min)/mL. The purified inulinase can be used in the production of high fructose syrups.

Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth.

UNLABELLED: L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD+-dependent L-lactate dehydrogenase. Naturally, Escherichia coli does not produce L-lactate in noticeable amounts, but can catabolize it via a dehydrogenation reaction mediated by an FMN-dependent L-lactate dehydrogenase. In aims to make the E. coli strain to produce L-lactate, three L-lactate dehydrogenase genes from different bacteria were cloned and expressed. The L-lactate producing strains, 090B1 (B0013-070, ΔldhA::diflldD::Pldh-ldhLca), 090B2 (B0013-070, ΔldhA::diflldD::Pldh-ldhStrb) and 090B3 (B0013-070, ΔldhA::diflldD::Pldh-ldhBcoa) were developed from a previously developed D-lactate over-producing strain, E. coli strain B0013-070 (ack-ptappspflBdldpoxBadhEfrdA) by: (1) deleting ldhA to block D-lactate formation, (2) deleting lldD to block the conversion of L-lactate to pyruvate, and (3) expressing an L-lactate dehydrogenase (L-LDH) to convert pyruvate to L-lactate under the control of the ldhA promoter. Fermentation tests were carried out in a shaking flask and in a 25-l bioreactor. Strains 090B1, 090B2 or 090B3 were shown to metabolize glucose to L-lactate instead of D-lactate. However, L-lactate yield and cell growth rates were significantly different among the metabolically engineered strains which can be attributed to a variation between temperature optimum for cell growth and temperature optimum for enzymatic activity of individual L-LDH. In a temperature-shifting fermentation process (cells grown at 37°C and L-lactate formed at 42°C), E. coli 090B3 was able to produce 142.2 g/l of L-lactate with no more than 1.2 g/l of by-products (mainly acetate, pyruvate and succinate) accumulated. In conclusion, the production of lactate by E. coli is limited by the competition relationship between cell growth and lactate synthesis. Enzymatic properties, especially the thermodynamics of an L-LDH can be effectively used as a factor to regulate a metabolic pathway and its metabolic flux for efficient L-lactate production. HIGHLIGHTS: The enzymatic thermodynamics was used as a tool for metabolic regulation. Minimizing the activity of L-lactate dehydrogenase in growth phase improved biomass accumulation. Maximizing the activity of L-lactate dehydrogenase improved lactate productivity in production phase.

Responding to the challenge of leprosy-related disability and ultra-poverty.

INTRODUCTION: The Millennium Development Goals have provided much needed attention to extreme poverty reduction. However, people with disabilities are disproportionately affected by poverty and in some countries, even the goal of US$1 per day is far out of reach. For people with leprosy-related disability living in ultra-poverty (on less than 50 cents a day), many mainstream poverty reduction strategies are inaccessible and inappropriate. METHOD: A project in north-west Bangladesh developed a more contextually meaningful definition of ultra-poverty according to nutrition energy intake. A total of 2372 people with leprosy-related disability were surveyed. Of those, 1285 individuals fell below the ultra-poverty line. Individualised interventions were implemented over an extended period of time, comprised of targeted practical assistance, enhancing community links, advocacy for entitlements, and further linking with other initiatives. RESULTS: Follow-up data available for 856 individuals showed an average increase in per capita income of 83%. Personal contribution to the family income increased by 65%. There was a 51% increase in families having access to a latrine. Finally families reported eating 30% more meals per day, up from an average of two meals per day. CONCLUSIONS: The initiative sought to address poverty in a wide variety of ways, using minimal inputs. Over several years, the results indicate a significant change in the economic situation of individuals with leprosy related disabilities. Other organisations are encouraged to duplicate the intervention and share their results.

Biodegradation of glycerol using bacterial isolates from soil under aerobic conditions.

Glycerol, a non-biodegradable by-product during biodiesel production is a major concern to the emerging biodiesel industry. Many microbes in natural environments have the ability to utilize glycerol as a sole carbon and energy source. The focus of this study was to screen for microorganisms from soil, capable of glycerol utilization and its conversion to value added products such as ethanol and 1,3-propanediol (1,3-PDO). Twelve bacterial isolates were screened for glycerol utilization ability in shake flask fermentations using M9 media supplemented with analytical grade glycerol (30 g/L) at various pH values (6, 7 and 8) and temperatures (30°C, 35°C and 40°C). Among these, six bacterial isolates (SM1, SM3, SM4, SM5, SM7 and SM8) with high glycerol degradation efficiency (>80%) were selected for further analysis. Highest level of 1,3-PDO production (15 g/L) was observed with isolate SM7 at pH 7 and 30°C, while superior ethanol production (14 g/L) was achieved by isolate SM9 at pH 8 and 35°C, at a glycerol concentration of 30 g/L. The selected strains were further evaluated for their bioconversion efficiency at elevated glycerol concentrations (50-110 g/L). Maximum 1,3-PDO production (46 g/L and 35 g/L) was achieved at a glycerol concentration of 70 g/L by isolates SM4 and SM7 respectively, with high glycerol degradation efficiency (>90). Three isolates (SM4, SM5 and SM7) also showed greater glycerol tolerance (up to 110 g/L). The isolates SM4 and SM7 were identified as Klebsiella pneumoniae and SM5 as Enterobacter aerogenes by 16S rDNA analysis. These novel isolates with greater glycerol tolerance could be used for the biodegradation of glycerol waste generated from the biodiesel industry into value-added commercial products.

A review on the diversity of antimicrobial peptides and genome mining strategies for their prediction.

Antibiotic resistance has become one of the most serious threats to human health in recent years. In response to the increasing microbial resistance to the antibiotics currently available, it is imperative to develop new antibiotics or explore new approaches to combat antibiotic resistance. Antimicrobial peptides (AMPs) have shown considerable promise in this regard, as the microbes develop low or no resistance against them. The discovery and development of AMPs still confront numerous obstacles such as finding a target, developing assays, and identifying hits and leads, which are time-consuming processes, making it difficult to reach the market. However, with the advent of genome mining, new antibiotics could be discovered efficiently using tools such as BAGEL, antiSMASH, RODEO, etc., providing hope for better treatment of diseases in the future. Computational methods used in genome mining automatically detect and annotate biosynthetic gene clusters in genomic data, making it a useful tool in natural product discovery. This review aims to shed light on the history, diversity, and mechanisms of action of AMPs and the data on new AMPs identified by traditional as well as genome mining strategies. It further substantiates the various phases of clinical trials for some AMPs, as well as an overview of genome mining databases and tools built expressly for AMP discovery. In light of the recent advancements, it is evident that targeted genome mining stands as a beacon of hope, offering immense potential to expedite the discovery of novel antimicrobials.

Beauveria Bassiana Amylase-Polygalacturonase Production Using Lignocellulosic Biomass and Application in Juice Processing.

Background: The search for sources of industrial biocatalysts, which are non-pathogenic and can utilise cheap nutrient sources, has been a continuous endeavour in the ~ 7 billion USD enzyme industry. Beauveria bassiana, an endophytic fungal entomopathogen, is non-pathogenic and possesses the potential to secrete various bioproducts while utilising readily available lignocellulosic biomass. Objective: This study investigated the optimised production of two glycosyl hydrolases, amylase and polygalacturonase, by B. bassiana while utilising readily available agricultural residues. Subsequently, the industrial potential of the enzymes in the clarification of fruit juice was evaluated. Materials and Methods: Initially, seven agro residues were screened for the concomitant production of amylase and polygalacturonase by B. bassiana SAN01. Subsequently, statistical optimisation tools, Plackett Burman Design (PBD) and Central Composite Design (CCD), were employed for the optimisation of enzyme production. The enzyme mixture was partially purified and applied in the clarification of pineapple juice. Result: The production of B. bassiana SAN01 amylase and polygalacturonase was found to be maximal while utilising wheat bran. Subsequent to PBD and CCD optimisation, the optimal conditions for enzyme production were identified to be at 30 °C, pH 6.0 and wheat bran concentration of ~40 g.L-1. Under these optimised conditions, heightened production levels of 34.82 and 51.05 U.mL-1 were recorded for amylase and polygalacturonase, respectively, which were 179% and 187% of the initial unoptimised levels. In addition, the most effective clarification of the juice (~90%) was observed at 35 °C after an incubation time of 120 min with no significant effect on the pH and total dissolved solids. Conclusion: B. bassiana, a well-known biocontrol agent, was shown to produce amylase and polygalacturonase using readily available agricultural residues for the first time. These enzyme production levels are the highest for these enzymes from any known endophytic fungal entomopathogen. This study further demonstrates the potential applicability of B. bassiana in other industrial processes besides its widespread use as a biopesticide.