Commercial Enzyme Products Research Articles

Transgenic Soybean for Production of Thermostable α-Amylase.

Alpha-amylases are crucial hydrolase enzymes which have been widely used in food, feed, fermentation, and pharmaceutical industries. Methods for low-cost production of α-amylases are highly desirable. Soybean seed, functioning as a bioreactor, offers an excellent platform for the mass production of recombinant proteins for its ability to synthesize substantial quantities of proteins. In this study, we generated and characterized transgenic soybeans expressing the α-amylase AmyS from Bacillus stearothermophilus. The α-amylase expression cassettes were constructed for seed specific expression by utilizing the promoters of three different soybean storage peptides and transformed into soybean via Agrobacterium-mediated transformation. The event with the highest amylase activity reached 601 U/mg of seed flour (one unit is defined as the amount of enzyme that generates 1 micromole reducing ends per min from starch at 65 °C in pH 5.5 sodium acetate buffer). The optimum pH, optimum temperature, and the enzymatic kinetics of the soybean expressed enzyme are similar to that of the E. coli expressed enzyme. However, the soybean expressed α-amylase is glycosylated, exhibiting enhanced thermostability and storage stability. Soybean AmyS retains over 80% activity after 100 min at 75 °C, and the transgenic seeds exhibit no significant activity loss after one year of storage at room temperature. The accumulated AmyS in the transgenic seeds represents approximately 15% of the total seed protein, or about 4% of the dry seed weight. The specific activity of the transgenic soybean seed flour is comparable to many commercial α-amylase enzyme products in current markets, suggesting that the soybean flour may be directly used for various applications without the need for extraction and purification.

Cellulase-assisted refining in a paperboard mill: Avoided emissions from energy savings. A case study of a Finnish paperboard mill

The 2023 being on its way to be the warmest year in the 174-year global surface temperature history it is becoming more and more evident how crucial it is to break the linkage between economic growth and greenhouse gas emissions.The pulp and paper industry is highly energy intensive industry sector. It is the fourth largest industrial energy user and currently produces approximately 1.3% of global greenhouse gas emissions. While the demand for paper and paperboard products keeps rising, it is essential for the industry to find energy efficient solutions for reducing the environmental impact of the pulp and paper products. 80% of the progress in energy efficiency in Finnish paper mills is due to improved technology and more optimal operational modes. Optimizing existing processes has bigger effect on energy efficiency than closing old and starting new paper mills.Pulp refining is one of the most energy intense steps in the paperboard production, representing 15%–18% of the total energy consumption of a paper mill. The effect of a commercial cellulase enzyme product was evaluated for energy savings and avoided emissions in the refining process of a paperboard mill. Data from a paperboard line using Bleached Softwood Kraft Pulp (BSKP) from a separate pulp mill was provided by a Finnish paperboard mill containing production runs both with and without commercial cellulase product ECOPULP® R. Data was collected from the mill automation system to evaluate and compare a refining capacity restricted run and a run that was not refining capacity restricted.Analysis of the data showed a reduction of refining energy by approximately 16%–17% when the cellulase enzyme was used. This corresponds to 2 kg of CO2 emissions avoided per tonne of pulp refined when calculated using the Finnish average electricity emissions intensity in 2022. Use of the cellulase enzyme enabled the refining results that could not have been reached with the bare mechanical refining capacity of the mill.Cellulases can be efficient processing aids in pulp refining. The reduction of required mechanical refining and electrical power to run the refiners to achieve the required freeness can significantly affect the carbon footprint of the paperboard products, whereas the negative impact of emissions from enzyme production is negligible. Reduction of CO2 emissions is especially significant in the areas where the electricity mix used for the production is based heavily on fossil fuels.

К вопросу о самообеспеченности страны молокосвертывающими ферментами

Food security is part of national security, self-sufficiency in basic foodstuffs being its important indicator. The situation with cheese production raw materials in the Russian Federation is gradually improving. According to the Ministry of Agriculture, the production of raw milk is growing, which means that the level of self-sufficiency is gradually increasing. However, milk-converting enzymes can create a bottleneck situation since the entire rennet cheese production depends on them. The share of imported milk-coagulating enzyme preparations on the Russian market is 84.3 %. Recombinant milk-converting enzymes dominate both domestic and global cheese-making. However, Russia has neither microbial recombinant chymosins nor coagulants of its own. The domestic commercial production of milk-converting enzymes, especially of modern recombinant preparations, is one of the most relevant issues the domestic food industry will have to tackle in the nearest future.

STABILITY PROPERTIES OF ALKALINE PROTEASES SOURCES AT DIFFERENT OPTIMUM CONDITIONS

– Alkaline proteases possess considerable industrial potential due to their biochemical diversity and wide applications in tannery and food industries, medicinal formulations, detergents and processes like waste treatment, silver recovery and resolution of amino acid mixtures. The genus Bacillus contains a number of industrially important species and approximately half of the present commercial production of bulk enzymes derives from the strains of Bacillus sp. These strains are specific producers of extracellular proteases and can be cultivated under extreme temperature and pH conditions to give rise to products that are, in turn, stable in a wide range of harsh environments.

Induction of the CtsR regulon improves Xylanase production in Bacillus subtilis

BackgroundThe bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome studies have been published that focus on enzyme secretion in B. subtilis. Here, we examined the effect of high-level expression of the commercially important enzyme endo-1,4-β-xylanase XynA on the B. subtilis transcriptome using RNA-seq.ResultsUsing the novel gene-set analysis tool GINtool, we found a reduced activity of the CtsR regulon when XynA was overproduced. This regulon comprises several protein chaperone genes, including clpC, clpE and clpX, and is controlled by transcriptional repression. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. When we abolished this negative feedback, by inactivating the repressor CtsR, the XynA production increased by 25%.ConclusionsOverproduction of enzymes can reduce the pool of Clp protein chaperones in B. subtilis, presumably due to negative feedback regulation. Breaking this feedback can improve enzyme production yields. Considering the conserved nature of Clp chaperones and their regulation, this method might benefit high-yield enzyme production in other organisms.

상업용 효소제를 이용한 무증자 쌀 발효 증류식 소주의 제조 가능성

The production of distilled soju by fermenting nonsteamed rice was evaluated using commercial enzyme products. White koji and modified nuruk had alpha-amylase activities of 31.90 U/g and 3,532.71 U/g, respectively, and gluco-amylase activities of 698.32 U/g and 4,899.58 U/g, respectively. The enzyme products had activities of 5,604.15-225,182.00 U/g and 13,517.41-120,822.41 U/g, respectively. At enzyme concentrations of >800 mg/L, the Chung-moo-purified enzyme had an alcohol productivity of ≥19%. Nurukzyme R400, Sanferm Yied, and Diazyme X4 exhibited alcohol productivities of >19% at concentrations of >600 mg/L. The alcohol content of the vacuum distillates was 41.31%-44.86%. The volatile component with the alcohol content adjusted to 25% was analyzed and principal component analysis was performed. The volatile components in white koji, Diazyme X4, and Sanferm Yield were similar. The modified nuruk treatment group had a relatively high ethyl lactate content compared to the white koji treatment group. The Nurukzyme R400 treatment group had high contents of butyric acid and ethyl butyrate. The Chung-moo-purified enzyme was characterized by a low component content. Thus, when enzyme products were used in nonsteamed rice fermentation, no effect on the alcohol productivity and quality of vacuum distilled soju was observed, suggesting that it can replace white koji and modified nuruk.

Enhanced energy savings in enzymatic refining of hardwood and softwood pulp

BackgroundPulp refining is an energy consuming, but integral part of paper production with the aim to increase tensile strength and smoothness of paper sheets. Commercial enzyme formulations are used to lower the energy requirements by pre-treatment of pulp before refining. However, a high number of different commercial enzyme products are available on the market containing enzymes of varying origin and composition, which complicates the prediction of their behavior, especially using different pulp types.ResultsEndoglucanase-rich enzyme formulations were characterized regarding enzyme activity at different temperatures, resulting in a significant decrease of activity above 70 °C. Some enzyme preparations additionally contained arabinosidase, xylanase and β-glucosidase activity consequently resulting in a release of xylose and glucose from pulp as determined by high-performance liquid chromatography. Interestingly, one enzyme formulation even showed lytic polysaccharide monooxygenase (LPMO) activity of 3.05 nkat mg−1. A correlation between enzyme activity using the endoglucanase specific derivatized cellopentaose (CellG5) substrate and enzyme performance in laboratory PFI (Papirindustriens forskningsinstitut) refining trials was observed on softwood pulp resulting in a maximum increase in the degree of refining values from 27.7°SR to 32.7°SR. When added to a purified endoglucanase enzyme (31.6°SR), synergistic effects were found for cellobiohydrolase II (34.7°SR) or β-glucosidase enzymes (35.7°SR) in laboratory refining. Comparison with previously obtained laboratory refining results on hardwood pulp allowed differences in enzyme performance based on varying pulp types to be elucidated.ConclusionsInterestingly, the individual enzymes indeed showed different refining effects on softwood and hardwood pulp. This difference could be predicted after development of an adapted enzyme activity assay by combination of the derivatized cellopentaose CellG5 substrate with either softwood or hardwood sulfate pulp.

Optimization of Crude Protease Production from Bacillus thuringiensis HSFI-12 and Thrombolytic Activity Its Enzyme Dialysate

Cardiovascular disease (CVD) is among the leading causes of death in the world caused by thrombosis. Thrombosis is the formation of excessive blood clots on the walls of blood vessels called thrombus. This could lead to fatal blockage of the heart muscle or brain. Thrombosis can be treated using enzyme-type drugs such as thrombolytic proteases. There have been many studies of bacteria producing thrombolytic enzymes isolated from fermented foodstuffs. However, commercial production of bacterial enzymes to fulfill the need of thrombolytic agents is still limited, causing high price of CVD therapy. Bacillus sp. HSFI-12 (Holothuria scabra Fermented Intestine-12) isolated from the fermented intestine of sea cucumber Holothuria scabra had been reported to have a competitive thrombolytic activity to the commercial Nattokinase. This study aimed to determine bacterial optimum incubation time based on enzyme activity after bacterium molecular identification was done. It also aimed to obtain the dialysate of bacterial crude protease and then determine the thrombolytic activity of the obtained dialysate. Thrombolytic activity was tested on 4 different types of blood (A, B, AB and O). Results of molecular identification showed that strain HSFI-12 shared similarity level of 99.80 %, with Bacillus thuringiensis. Activity of crude protease from the incubated cultures peaked at 48 h of incubation with activity of 191.5 U/mL The activity of concentrated protease after precipitation and dialysis process (dialysate) was 2-times higher by 355,7 U/mL. The percentage of lysis of blood clots produced by crude blood groups A, B, AB and O showed a range of values ​​of 66.423 - 67.656 % while those that produce dialysate are 77.564 - 78.861 %. As conclusion, the best (optimized) incubation time to produce crude enzyme from B. thuringiensis HSFI-12 with the highest activity was 48 h. The process of concentrating the crude thrombolytic protease of B. thuringiensis HSFI-12 increases both activity and thrombolytic ability of the bacterial enzyme. HIGHLIGHTS The most optimum incubation time for protease production from Bacillus thuringiensis is 48 h Partial purification on bacterial crude protease through resulted in 86 % increased activity Partial purification on bacterial crude protease resulted in 17 % increased blood clot lysis ability Clot lysis ability of both crude and dialysate proteases are higher than that of Nattokinase (NK) GRAPHICAL ABSTRACT

Biodegradation of Organic Waste Using Bacillus Species Isolated From Soil

Organic waste can be enzymatically degraded by microbes. In this study, the Bacillus species were isolated from soil and identified as Bacillus subtilis, Bacillus licheniformis, Bacillus macquariensis, Bacillus brevis, and Bacillus circulans which were optimized considering pH (5, 7, 9) and temperature (37°C, 45°C, 55°C) for the maximum production of amylase, gelatinase, lipase, and cellulase, principally for the degradation of organic waste. The maximum production of amylase was found at 37°C with pH 7 and 9, gelatinase and lipase at 37°C with pH 5,7,9 by almost all identified species. Similarly, the production of cellulase was shown by Bacillus licheniformis only at 45°C, pH 5. The degradation was confirmed by the analysis of the solid content of degraded waste. The maximum degradation of starch and lipid-containing waste was shown by Bacillus macquariensis whereas Bacillus circulans were able to degrade gelatin-containing waste effectively. Bacillus species showed a synergistic effect in biodegradation. Bacillus subtilis and Bacillus licheniformis used in ratios 1:1 and 1:2 were found to be effective degraders of lipid and starch-containing waste respectively. Bacillus macquariensis, Bacillus brevis, and Bacillus circulans used in ratio 1:1:1 showed effective degradation of gelatin-containing waste. The degradation of the organic waste by multi-enzyme producer Bacillus species can be the most effective and eco-friendly method and their optimization for enzyme production can be beneficial for commercial enzyme production as well as for biotechnological applications. Int. J. Appl. Sci. Biotechnol. Vol 10(2): 104-111.

Diversity of medicinal plants used for skincare by Bulungan tribe in North Kalimantan, Indonesia and its melanin biosynthesis inhibition

Abstract. Kartina, Batubara I, Kuspradini H, Kusuma IW, Wahyuni WT, Egra S, Matsunaga T. 2022. Diversity of medicinal plants used for skincare by Bulungan tribe in North Kalimantan, Indonesia and its melanin biosynthesis inhibition. Biodiversitas 23: 1246-1253. Indigenous people of the Bulungan tribe in North Kalimantan, Indonesia have been utilizing plants for health purposes, including for skincare such as preventing skin from being dark due to melanin production. This study aimed to evaluate the diversity of medicinal plants utilized by the Bulungan tribe for skincare and determine the melanin biosynthesis inhibition activity. Plants having medicinal uses were investigated through literature studies and interviews with the members of Bulungan tribe in Tanjung Palas Tengah Village. Melanin biosynthesis inhibition was determined by identifying the tyrosinase inhibition potential using commercial tyrosinase enzyme, while intracellular and extracellular melanin biosynthesis production activity was determined using B16 melanoma cells. The results showed at least 19 plant species that have medicinal uses, including for skincare, with some of them having the potential for melanogenesis, both in suppressing and producing melanin. We found a plant sample with melanin suppression potential on B16 melanoma cell, namely Jatropha curcas, at 6.2 µg/ml concentration, which could decrease extracellular melanin content by 18%. Tyrosinase activity with L-tyrosine showed that Cinnamomum burmannii (root) (IC50: 3.4 ?g/mL) showed an insignificant difference with kojic acid, which served as positive control by showing an inhibitory level of (IC50: 0.14 ?g/mL). Based on the data obtained, a pharmacological study is recommended to investigate melanin biosynthesis as a skin whitening agent. We believe that this study can become basic data to discover several active compounds in future studies.

Detection and diversity of the mannosylerythritol lipid (MEL) gene cluster and lipase A and B genes of Moesziomyces antarcticus isolated from terrestrial sites chronically contaminated with crude oil in Trinidad

BackgroundMannosylerythritol lipids (MELs) belong to the class of glycolipid biosurfactants and are produced by members of the Ustilago and Moesziomyces genera. Production of MELs is regulated by a biosynthetic gene cluster (MEL BGC). Extracellular lipase activity is also associated with MEL production. Most microbial glycolipid-producers are isolated from oil-contaminated environments. MEL-producing yeast that are capable of metabolizing crude oil are understudied, and there is very limited data on indigenous strains from tropical climates. Analysis of the MEL BGC and lipase genes in Trinidad M. antarcticus strains, using a gene-targeted approach, revealed a correlation between their intrinsic capability to degrade crude oil and their adaptation to survive in a chronically polluted terrestrial environment.ResultsM. antarcticus was isolated from naturally-occurring crude oil seeps and an asphaltic mud volcano in Trinidad; these are habitats that have not been previously reported for this species. Genus identification was confirmed by the large-subunit (LSU) and the small-subunit (SSU) sequence comparisons and species identification was confirmed by ITS sequence comparisons and phylogenetic inference. The essential genes (Emt1, Mac1, Mac2, Mmf1) of the MEL BGC were detected with gene-specific primers. Emt1p, Mac1p and Mmf1p sequence analyses confirmed that the Trinidad strains harboured novel synonymous amino acid (aa) substitutions and structural comparisons revealed different regions of disorder, specifically for the Emt1p sequence. Functionality of each protein sequence was confirmed through motif mining and mutation prediction. Phylogenetic relatedness was inferred for Emt1p, Mac1p and Mmf1p sequences. The Trinidad strains clustered with other M. antarcticus sequences, however, the representative Trinidad M. antarcticus sequences consistently formed a separate, highly supported branch for each protein. Similar phylogenetic placement was indicated for LipA and LipB nucleotide and protein sequences. The Trinidad strains also demonstrated lipolytic activity in culture, with an ability to utilize different carbon sources. Comparative evolution of MEL BGC and LipA gene suggested early and late duplication events, depending on the gene, followed by a number of speciation events within Ustilaginaceae. M. antarcticus and M. aphidis were separated from all other members of Ustilaginaceae and two gene homologues were detected, one for each species.ConclusionsSequence analyses was based on a novel gene-targeted approach to analyze the essential genes of the MEL BGC and LipA and LipB genes of M. antarcticus strains from Trinidad. The findings indicated that these strains accumulated nucleotide mutations to a threshold level that did not affect the function of specific proteins encoded by the MEL BGC and LipA and LipB genes. The biosurfactant and lipase enzymes secreted by these Trinidad M. antarcticus strains facilitated their survival in oil-contaminated terrestrial environments. These findings suggest that the Trinidad strains should be explored as promising candidates for the commercial production of MEL biosurfactants and lipase enzymes.

Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain.

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

Characterization of Genetically Modified Microorganisms Using Short- and Long-Read Whole-Genome Sequencing Reveals Contaminations of Related Origin in Multiple Commercial Food Enzyme Products.

Despite their presence being unauthorized on the European market, contaminations with genetically modified (GM) microorganisms have repeatedly been reported in diverse commercial microbial fermentation produce types. Several of these contaminations are related to a GM Bacillus velezensis used to synthesize a food enzyme protease, for which genomic characterization remains currently incomplete, and it is unknown whether these contaminations have a common origin. In this study, GM B. velezensis isolates from multiple food enzyme products were characterized by short- and long-read whole-genome sequencing (WGS), demonstrating that they harbor a free recombinant pUB110-derived plasmid carrying antimicrobial resistance genes. Additionally, single-nucleotide polymorphism (SNP) and whole-genome based comparative analyses showed that the isolates likely originate from the same parental GM strain. This study highlights the added value of a hybrid WGS approach for accurate genomic characterization of GMM (e.g., genomic location of the transgenic construct), and of SNP-based phylogenomic analysis for source-tracking of GMM.

Recombinant production and characterisation of two chitinases from Rasamsonia emersonii, and assessment of their potential industrial applicability.

Rasamsonia emersonii (previously Talaromyces emersonii) is a thermophilic filamentous fungus displaying optimum growth at 45°C. It has a history of use in commercial food enzyme production. Its unfractionated chitinolytic secretome was partially characterised in the early 1990s; however, no individual chitinase from this source has been described in literature previously. This study describes two GH18 chitinases originating from the R. emersonii genome, expressed in the methylotrophic yeast P. pastoris. Chit1 comprises of a GH18 catalytic domain and Chit2 comprises of a GH18 catalytic domain and a chitin-binding motif at the C-terminal. The chitinases were expressed as glycoproteins. The apparent molecular weight of Chit1 was 35.8-42.1kDa with a smearing tail associated with glyco-sidechains visible up to 72.2kDa. This became two bands of 30.8 and 29.0kDa upon de-glycosylation. The apparent molecular weight of Chit2 was 50.4kDa, reducing to 48.2kDa upon de-glycosylation. Both chitinases displayed endo-chitinase and chitobiosidase activity, temperature optima of 50-55°C and low pH optima (pH 4.5 or lower); Chit1 displayed a pH optimum of 3.5, retaining > 60% maximum activity at pH 2.2, a pH range lower than most enzymes of fungal origin. Chit2 displayed the highest chitin-degrading ability at 3456µmol/mg on 4-NP-triacetylchitotriose, but lost activity faster than Chit1, which displayed 403µmol/mg on the same substrate. The predicted D values (time required to reduce the enzyme activity to 10% of its original value at 50°C) were 19.2 and 2.3days for Chit1 and Chit2, respectively. Thus, Chit1 can be considered one of few hyperthermostable chitinase enzymes described in literature to date. Their physicochemical properties render these chitinases likely suitable for shrimp chitin processing including one-step chitin hydrolysis and alternative sustainable protein processing and the attractive emerging application of mushroom food waste valorisation.Key points• Two GH18 chitinases originating from the industrially relevant thermophilic fungus R. emersonii were cloned and expressed in P. pastoris.• The purified recombinant chitinases showed low pH and high temperature optima and appreciable thermostability at industrially relevant temperatures.• The chitinases displayed characteristics that indicate their likely suitability to several industrial applications including sustainable alternative protein processing, food waste valorisation of commercial mushroom production and one-step shrimp chitin processing.

Magnetophoretic and spectral characterization of oxyhemoglobin and deoxyhemoglobin: Chemical versus enzymatic processes.

A new method for hemoglobin (Hb) deoxygenation, in suspension or within red blood cells (RBCs) is described using the commercial enzyme product, EC-Oxyrase®. The enzymatic deoxygenation method has several advantages over established deoxygenation methodologies, such as avoiding side reactions that produce methemoglobin (metHb), thus eliminating the need for an inert deoxygenation gas and airtight vessel, and facilitates easy re-oxygenation of Hb/RBCs by washing with a buffer that contains dissolved oxygen (DO). The UV-visible spectra of deoxyHb and metHb purified from human RBCs using three different preparation methods (sodium dithionite [to produce deoxyHb], sodium nitrite [to produce metHb], and EC-Oxyrase® [to produce deoxyHb]) show the high purity of deoxyHb prepared using EC-Oxyrase® (with little to no metHb or hemichrome production from side reactions). The oxyHb deoxygenation time course of EC-Oxyrase® follows first order reaction kinetics. The paramagnetic characteristics of intracellular Hb in RBCs were compared using Cell Tracking Velocimetry (CTV) for healthy and sickle cell disease (SCD) donors and oxygen equilibrium curves show that the function of healthy RBCs is unchanged after EC-Oxyrase® treatment. The results confirm that this enzymatic approach to deoxygenation produces pure deoxyHb, can be re-oxygenated easily, prepared aerobically and has similar paramagnetic mobility to existing methods of producing deoxyHb and metHb.

THE ROLES OF ENZYME IN FOOD PROCESSING - AN OVERVIEW

Enzymes haven used long in food processing before their discovery as a biological catalyst. Food fermentation was among the early art of food processing and the use of enzymes in fermentation and cheese making started about 6000 BC. The roles of enzymes in food processing and preservation contributed to the development of mankind. They contributed in the areas of baking, cheese making, dairy processing, milling, cereals technology, juice and beverages processing, vegetable processing, oils and fats processing, and wine processing among others. Microorganisms are the earliest and foremost source of enzymes used in food processing, other sources are plant and animal tissues and organs. Advances in science and technology disclosed more potentials of enzymes and biotechnology open doors for commercial production of enzymes with charming properties. The development of enzyme immobilization techniques allows the reused of enzymes without affecting their properties, structure, or activities. Recent advances in genetic engineering and recombinant DNA technology permit the production of enzymes with exceptional properties. The current trends in the production of Extremozymes will open doors for using enzymes under extreme conditions of temperature, pH, and pressure. In food, processing enzymes can be used as ingredients, processing aid, or as a catalyst for both pre-and post-consumption catalysis. Enzymes improve the quality, shelf life, stability, and sensory properties of foods. They play important roles in food processing by lowering energy consumption, minimizing waste, producing desired products specifically required, and making foods more affordable, palatable, and available

Fungal interactions induce changes in hyphal morphology and enzyme production

ABSTRACT In nature, species interacts/competes with one other within their surrounding for food and space and the type of interactions are unique to each species. The interacting partners secrete different metabolites, which may have high importance in human welfare. Fungal–fungal interactions are complex mechanisms that need better understanding. Here, 14 fungal isolates were facilitated in 105 possible combinations to interact on potato dextrose agar. Morphologically, no changes were observed when the same fungal isolates were allowed to interact within them. However, 10 interactions between different fungal isolates showed mutual replacement with each fungus; capturing territory from the other. Contrastingly, 35 interactions resulted into complete replacement as one of the fungi was inhibited by rapid growth of the other fungus. In 46 interactions, formation of barrage was observed leading to deadlock type of interaction wherein both fungi have restricted growth. To study in details about the barrage formation, two fungal interactions were taken (i) T. coccinea vs. L. lactinea and (ii) T. coccinea vs. T. versicolor. Microscopic changes in the hyphal growth during interaction were observed. There was significant increase in the enzymatic activities including cellulase, xylanase and chitinase during in-vitro fungal–fungal interaction, suggesting the importance of such interactions for commercial enzyme production.

Optimization and scale-up of \u03b1-amylase production by Aspergillus oryzae using solid-state fermentation of edible oil cakes

BackgroundAmylases produced by fungi during solid-state fermentation are the most widely used commercial enzymes to meet the ever-increasing demands of the global enzyme market. The use of low-cost substrates to curtail the production cost and reuse solid wastes are seen as viable options for the commercial production of many enzymes. Applications of α-amylases in food, feed, and industrial sectors have increased over the years. Additionally, the demand for processed and ready-to-eat food has increased because of the rapid growth of food-processing industries in developing economies. These factors significantly contribute to the global enzyme market. It is estimated that by the end of 2024, the global α-amylase market would reach USD 320.1 million (Grand View Research Inc., 2016). We produced α-amylase using Aspergillus oryzae and low-cost substrates obtained from edible oil cake, such as groundnut oil cake (GOC), coconut oil cake (COC), sesame oil cake (SOC) by solid-state fermentation. We cultivated the fungus using these nutrient-rich substrates to produce the enzyme. The enzyme was extracted, partially purified, and tested for pH and temperature stability. The effect of pH, incubation period and temperature on α-amylase production using A. oryzae was optimized. Box–Behnken design (BBD) of response surface methodology (RSM) was used to optimize and determine the effects of all process parameters on α-amylase production. The overall cost economics of α-amylase production using a pilot-scale fermenter was also studied.ResultsThe substrate optimization for α-amylase production by the Box–Behnken design of RSM showed GOC as the most suitable substrate for A. oryzae, as evident from its maximum α-amylase production of 9868.12 U/gds. Further optimization of process parameters showed that the initial moisture content of 64%, pH of 4.5, incubation period of 108 h, and temperature of 32.5 °C are optimum conditions for α-amylase production. The production increased by 11.4% (10,994.74 U/gds) by up-scaling and using optimized conditions in a pilot-scale fermenter. The partially purified α-amylase exhibited maximum stability at a pH of 6.0 and a temperature of 55 °C. The overall cost economic studies showed that the partially purified α-amylase could be produced at the rate of Rs. 622/L.ConclusionsThe process parameters for enhanced α-amylase secretion were analyzed using 3D contour plots by RSM, which showed that contour lines were more oriented toward incubation temperature and pH, having a significant effect (p < 0.05) on the α-amylase activity. The optimized parameters were subsequently employed in a 600 L-pilot-scale fermenter for the α-amylase production. The substrates were rich in nutrients, and supplementation of nutrients was not required. Thus, we have suggested an economically viable process of α-amylase production using a pilot-scale fermenter.

Microbial alkaline serine proteases: Production, properties and applications.

Proteolytic enzymes hold a pivotal position in numerous industrial processes where hydrolysis of protein molecules is required under precise conditions. The emerging trend of biotechnological applications in recent years has witnessed a renewed interest in alkaline serine proteases extending their utility in detergent, leather, textile, food and pharmaceutical industries. A variety of microorganisms have been reported to produce alkaline serine proteases on a large scale, however, extensive research to find an alkaline serine protease with desirable characteristics such as significant catalytic efficiency, expanded stability and broad substrate specificity is still ongoing. Although submerged fermentation dominates the commercial enzyme production, recent reports have emphasized on solid state fermentation technology which can reduce major cost associated with the enzyme production. In the present review, recent research on alkaline serine proteases along with their novel properties and production using solid state fermentation have been discussed.

육계에서 에너지 및 조단백질 저감 사료 내 효소제의 적용 효과

This study investigated the effects of the addition of commercial enzyme products to broiler feed with reduced levels of metabolizable energy and crude protein on the growth performance, blood profiles, and cecal microflora. A total of 750 seven-day-old male Ross 308 broiler chicks were randomly allotted to five treatment groups with six replicates (25 birds/replicate) for 28 days. A corn-wheat-soybean meal-based diet was formulated to meet or exceed the nutrient recommendations and used as the control diet. Experimental diets with metabolizable energy reduced by 100 kcal/kg and crude protein by 1% were formulated containing four different commercial enzyme products. Feed intake and weight gain were not affected by the dietary treatments; however, feed conversion ratios were significantly lower (<i>P</i>&lt;0.01) in the control group than in the other treatment groups during the entire experimental period. There were no significant differences in the blood profiles and cecal microflora between the dietary treatments. However, the viscosity of jejunal contents in chicks fed a diet with enzyme B was significantly lower than that in the other groups. The tentative marketing age was unaffected by any of the dietary treatments, and ranged from 26.39 d to 26.84 d. Collectively, the use of commercial enzymes contributed to a similar weight gain as that in chicks fed diets with reduced energy and crude protein levels; however, it failed to maintain the feed conversion ratio.