Cost Of Enzyme Production Research Articles

Novel Approach of Tackling Wax Deposition Problems in Pipeline Using Enzymatic Degradation Process: Challenges and Potential Solutions

Anthropogenic activities have led to hydrocarbon spills, and while traditional bioremediation methods are costly and time-consuming, recent research has focused on engineered enzymes for managing pollutant. The potential of enzymes for resolving wax flow problems in the petroleum industry remains unexplored. This paper offers a comprehensive review of the current state of research activities related to the bioremediation of petroleum-polluted sites and the biodegradation of specific petroleum hydrocarbons. The assayed enzymes that took part in the degradation were discussed in detail. Lipase, laccase, alkane hydroxylase, alcohol dehydrogenase, esterase, AlkB homologs and cytochrome P450 monooxygenase are among the enzymes responsible for the degradation of more than 50% of the hydrocarbons in contaminated soil and wastewater and found to be active on carbon C8 to C40. The possible biodegradation mechanism of petroleum hydrocarbons was also elucidated. The enzymes’ primary metabolic pathways include terminal, subterminal, and ω-oxidation. Next, given the successful evidence of the hydrocarbon treatment efficiency, the authors analyzed the opportunity for the enzymatic degradation approach if it were to be applied to a different scenario: managing wax deposition in petroleum-production lines. With properties such as high transformation efficiency and high specificity, enzymes can be utilized for the treatment of viscous heavy oil for transportability, evidenced by the 20 to 99% removal of hydrocarbons. The challenges associated with the new approach are also discussed. The production cost of enzymes, the characteristics of hydrocarbons and the operating conditions of the production line may affect the biocatalysis reaction to some extent. However, the challenges can be overcome by the usage of extremophilic enzymes. The combination of technological advancement and deployment strategies such as the immobilization of a consortium of highly thermophilic and halotolerant enzymes is suggested. Recovering and reusing enzymes offers an excellent strategy to improve the economics of the technology. This paper provides insights into the opportunity for the enzymatic degradation approach to be expanded for wax deposition problems in pipelines.

OPTIMIZATION OF CARBOXY METHYL CELLULASE PRODUCTION BY CLADOSPORIUM SP. WITH FOOD WASTE IN BAGASSE SOLID STATE FERMENTATION USING RESPONSE SURFACE METHODOLOGY

Enzyme production costs should be reduced by using inexpensively available substrates. In this, we determine the effects of various medium components and food processing waste on Cladosporium sp. production of CMCase in SSF. Five factors were tested with a three-level Box-Wilson factorial, central composite design (CCD) to determine how they impacted Cladosporium sp. NCIM 901's ability to produce CMCase. For the model, the R2 (coefficient of determination) value was 0.9349. Based on the process findings, the optimal medium for higher enzyme synthesis had wheat bran (4 g/L), cacl2 (2 g/L), urea (7.5 g/L) and rice bran (8.76 g/L), with the medium's original pH at 5.5. The model's accuracy is confirmed by the experimental result of CMCase, which is 23.42 U/gds and equivalent to the expected yield of 22.31 U/gds. The accuracy of the current experimental design has been verified by experimental validation. The enzyme's properties indicated that its greatest activity occurred at pH 5.5 and that its ideal temperature was about 50 °C. The enzyme is more active toward CMC and less active toward filter paper. CaCl2 and MgCl2 increased the enzyme's activity. Enzymatic hydrolysis of waste-derived polysaccharides into simple sugars is necessary for the industrial manufacture of bioethanol. The efficacy of the produced Cladosporium sp. cellulase (CMCase) enzyme to saccharify pearl millet grass results shows that H2SO4 treated substrate gave more fermentable sugars compared to the untreated substrate. Results suggest that the indigenous produced-CMCase can be efficiently used for lignocellulosic biomass saccharification (55.40%) compared to the untreated sample (37.10%) in 48 h, which will eventually reduce the cost utilized for purchasing commercial enzymes.

Fungal endo and exochitinase production, characterization, and application for Candida biofilm removal.

Chitinases are promising enzymes for a multitude of applications, including chitooligosaccharide (COS) synthesis for food and pharmaceutical uses and marine waste management. Owing to fungal diversity, fungal chitinases may offer alternatives for chitin degradation and industrial applications. The rapid reproduction cycle, inexpensive growth media, and ease of handling of fungi may also contribute to reducing enzyme production costs. Thus, this study aimed to identify fungal species with chitinolytic potential and optimize chitinase production by submerged culture and enzyme characterization using shrimp chitin. Three fungal species, Coriolopsis byrsina, Trichoderma reesei, and Trichoderma harzianum, were selected for chitinase production. The highest endochitinase production was achieved in C. byrsina after 168h cultivation (0.3 U mL- 1). The optimal temperature for enzyme activity was similar for the three fungal species (up to 45 and 55 ºC for endochitinases and exochitinases, respectively). The effect of pH on activity indicated maximum hydrolysis in acidic pH (4-7). In addition, the crude T. reesei extract showed promising properties for removing Candida albicans biofilms. This study showed the possibility of using shrimp chitin to induce chitinase production and enzymes that can be applied in different industrial sectors.

Optimization Production of an Endo-β-1,4-Xylanase from Streptomyces thermocarboxydus Using Wheat Bran as Sole Carbon Source

Xylanases, key enzymes for hydrolyzing xylan, have diverse industrial applications. The bioprocessing of agricultural byproducts to produce xylanase through fermentation approaches is gaining importance due to its significant potential to reduce enzyme production costs. In this work, the productivity of Streptomyces thermocarboxydus TKU045 xylanase was enhanced through liquid fermentation employing wheat bran as the sole carbon source. The maximum xylanase activity (25.314 ± 1.635 U/mL) was obtained using the following optima factors: 2% (w/v) wheat bran, 1.4% (w/v) KNO3, an initial pH of 9.8, an incubation temperature of 37.3 °C, and an incubation time of 2.2 days. Xylanase (Xyn_TKU045) of 43 kDa molecular weight was isolated from the culture supernatant and was biochemically characterized. Analysis through liquid chromatography with tandem mass spectrometry revealed a maximum amino acid identity of 19% with an endo-1,4-β-xylanase produced by Streptomyces lividans. Xyn_TKU045 exhibited optimal activity at pH 6, with remarkable stability within the pH range of 6.0 to 8.0. The enzyme demonstrated maximum efficiency at 60 °C and considerable stability at ≤70 °C. Mg2+, Mn2+, Ba2+, Ca2+, 2-mercaptoethanol, Tween 20, Tween 40, and Triton X-100 positively influenced Xyn_TKU045, while Zn2+, Fe2+, Fe3+, Cu2+, and sodium dodecyl sulfate exhibited adverse impact. The kinetic properties of Xyn_TKU045 were a Km of 0.628 mg/mL, a kcat of 75.075 s−1 and a kcat/Km of 119.617 mL mg−1s−1. Finally, Xyn_TKU045 could effectively catalyze birchwood xylan into xylotriose and xylobiose as the major products.

Graphene and its derivatives fabrication from paddy straw for improved and sustainable application in biofuels production: New Insight

One of the most sustainable processes for producing green fuels for industrial use is biomass to biofuels production. However, the high production cost of enzymes, the low functional stability of microbes and enzymes with low sugar, and fuel production are key challenges. Nanocatalyst implementation can be a potential strategy to resolve the technical hurdles of biomass-based biofuel production. Due to its distinct physicochemical features, graphene and its derivatives have recently attracted a lot of interest in microbial interaction and microbial based biomass to biofuels production technologies. The diverse physicochemical properties of graphene have created new prospects for biomass to biofuels manufacturing technology to become more cost-effective at the pilot stage. However, sustainable fabrication methods and scale-up process optimization are still not well explored for mass application trials. Therefore, the present review is based on developing a sustainable approach towards the green synthesis of graphene and its derivatives from paddy straw waste. Furthermore, potential opportunities for low-cost synthesis approaches of graphene and its derivatives from paddy straw and their sustainable application in economical biomass to biofuel production processes using paddy straw as a substrate itself, as well as current challenges and feasible future applications, have been discussed.

A thermostable organic solvent-tolerant lipase from Brevibacillus sp.: production and integrated downstream processing using an alcohol-salt-based aqueous two-phase system.

Lipases are used for the synthesis of different compounds in the chemical, pharmaceutical, and food industries. Most of the reactions are carried out in non-aqueous media and often at elevated temperature, requiring the use of organic solvent-tolerant thermostable lipases. However, most known lipases are not stable in the presence of organic solvents and at elevated temperature. In this study, an organic solvent-tolerant thermostable lipase was obtained from Brevibacillus sp. SHI-160, a moderate thermophile isolated from a hot spring in the East African Rift Valley. The enzyme was optimally active at 65°C and retained over 90% of its activity after 1 h of incubation at 70°C. High lipase activity was measured in the pH range of 6.5 to 9.0 with an optimum pH of 8.5. The enzyme was stable in the presence of both polar and non-polar organic solvents. The stability of the enzyme in the presence of polar organic solvents allowed the development of an efficient downstream processing using an alcohol-salt-based aqueous two-phase system (ATPS). Thus, in the presence of 2% salt, over 98% of the enzyme partitioned to the alcohol phase. The ATPS-recovered enzyme was directly immobilized on a solid support through adsorption and successfully used to catalyze a transesterification reaction between paranitrophenyl palmitate and short-chain alcohols in non-aqueous media. This shows the potential of lipase SHI-160 to catalyze reactions in non-aqueous media for the synthesis of valuable compounds. The integrated approach developed for enzyme production and cheap and efficient downstream processing using ATPS could allow a significant reduction in enzyme production costs. The results also show the potential of extreme environments in the East African Rift Valley as sources of valuable microbial genetic resources for the isolation of novel lipases and other industrially important enzymes.

Enzyme selection, optimization, and production toward biodegradation of post-consumer poly(ethylene terephthalate) at scale.

Poly(ethylene terephthalate) (PET) is one of the world's most widely used polyester plastics. Due to its chemical stability, PET is extremely difficult to hydrolyze in a natural environment. Recent discoveries in new polyester hydrolases and breakthroughs in enzyme engineering strategies have inspired enormous research on biorecycling of PET. This study summarizes our research efforts toward large-scale, efficient, and economical biodegradation of post-consumer waste PET, including PET hydrolase selection and optimization, high-yield enzyme production, and high-capacity enzymatic degradation of post-consumer waste PET. First, genes encoding PETase and MHETase from Ideonella sakaiensisand the ICCG variant of leaf-branch compost cutinase (LCCICCG ) were codon-optimized and expressed in Escherichia coliBL21(DE3) for high-yield production. To further lower the enzyme production cost, a pelB leader sequence was fused to LCCICCG so that the enzyme can be secreted into the medium to facilitate recovery. To help bind the enzyme on the hydrophobic surface of PET, a substrate-binding module in a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM) was fused to the C-terminus of LCCICCG . The resulting four different LCCICCG variants (LCC, PelB-LCC, LCC-PBM, and PelB-LCC-PBM), together with PETase and MHETase, were compared for PET degradation efficiency. A fed-batch fermentation process was developed to produce the target enzymes up to 1.2g/L. Finally, the best enzyme, PelB-LCC, was selected and used for the efficient degradation of 200g/L recycled PET in a well-controlled, stirred-tank reactor. The results will help develop an economical and scalable biorecycling process toward a circular PET economy. This article is protected by copyright. All rights reserved.

Immobilization of Cellulolytic Enzymes in Accurel® MP1000

Cellulases are a class of enzymes of great industrial interest that present several strategic applications. However, the high cost of enzyme production, coupled with the instabilities and complexities of proteins required for hydrolytic processes, still limits their use in several protocols. Therefore, enzyme immobilization may be an essential tool to overcome these issues. The present work aimed to evaluate the immobilization of cellulolytic enzymes of the commercial enzyme cocktail Celluclast® 1.5 L in comparison to the cellulolytic enzyme cocktail produced from the wild strain Trichoderma harzianum I14-12 in Accurel® MP1000. Among the variables studied were temperature at 40 °C, ionic strength of 50 mM, and 72 h of immobilization, with 15 m·L −1 of proteins generated biocatalysts with high immobilization efficiencies (87% for ACC-Celluclast biocatalyst and 95% for ACC-ThI1412 biocatalyst), high retention of activity, and specific activities in the support for CMCase (DNS method), FPase (filter paper method) and β-glucosidase (p-nitrophenyl-β-D-glucopyranoside method). Presenting a lower protein concentration (0.32 m·L−1) than the commercial Celluclast® 1.5 L preparation (45 m·L−1), the ACC-ThI1412-derived immobilized biocatalyst showed thermal stability at temperatures higher than 60 °C, maintaining more than 90% of the residual activities of FPase, CMCase, and β-glucosidase. In contrast, the commercial-free enzyme presented a maximum catalytic activity at only 40 °C. Moreover, the difference in molecular weight between the component enzymes of the extract was responsible for different hydrophobic and lodging interactions of proteins on the support, generating a robust and competitive biocatalyst.

Validation of mathematical models as a tool for prediction of α-amylase production by Coprinus comatus in a low-cost culture medium

Studying new microbial sources and evaluating the growth kinetics behavior to predict large-scale production become essential. Besides, research with low-cost culture media has gained interest due to the need to decrease enzyme production costs. The literature concerning the use of macromycetes as a source of amylases, and applying mathematical models to predict their behavior is scarce. In this study, four growth kinetics in different compositions of culture media were carried out: two of them using different concentrations of Low-Grade Flour (LGF), one wheat mill by-product and two synthetic ones. In the kinetics mathematical models were used to evaluate the bioprocesses and their scaling feasibility. Mathematical models, such as modified Logistic, modified Gompertz, Baranyi, and Roberts, and modified Luedeking-Piret, were applied to predict mycelial growth, enzyme production, and substrate consumption. The results showed the model’s capability to predict mycelial growth (biomass) and substrate consumption (starch concentration). On the other hand, the mathematical models efficiently describe only the maximum enzymatic activity (α-amylase), not the process's other parameters, because of the complexity and diversity of biomolecules synthesized by Coprinus comatus. The wheat mill by-product was efficient as an ingredient in the culture media composition. Furthermore, it was concluded that B2 formulation (culture medium with 48.5 g/L of LGF) is the batch with the highest potential for the α-amylase production from C. comatus and the expansion of this process on an industrial scale. This study is important and valid for the scientific community since modeling studies for macromycetes are scarce in the literature.

CAZyme from gut microbiome for efficient lignocellulose degradation and biofuel production

Over-exploitation and energy security concerns of the diminishing fossil fuels is a challenge to the present global economy. Further, the negative impact of greenhouse gases released using conventional fuels has led to the need for searching for alternative biofuel sources with biomass in the form of lignocellulose coming up as among the potent candidates. The entrapped carbon source of the lignocellulose has multiple applications other than biofuel generation under the biorefinery approach. However, the major bottleneck in using lignocellulose for biofuel production is its recalcitrant nature. Carbohydrate Active Enzymes (CAZymes) are enzymes that are employed for the disintegration and consumption of lignocellulose biomass as the carbon source for the production of biofuels and bio-derivatives. However, the cost of enzyme production and their stability and catalytic efficiency under stressed conditions is a concern that hinders large-scale biofuel production and utilization. Search for novel CAZymes with superior activity and stability under industrial condition has become a major research focus in this area considering the fact that the most conventional CAZymes has low commercial viability. The gut of plant-eating herbivores and other organisms is a potential source of CAZyme with high efficiency. The review explores the potential of the gut microbiome of various organisms in the production of an efficient CAZyme system and the challenges in using the biofuels produced through this approach as an alternative to conventional biofuels.

Immobilization of Recombinant Endoglucanase (CelA) from Clostridium thermocellum on Modified Regenerated Cellulose Membrane

Cellulases are being widely employed in lignocellulosic biorefineries for the sustainable production of value-added bioproducts. However, the high production cost, sensitivity, and non-reusability of free cellulase enzymes impede their commercial applications. Enzyme immobilization seems to be a potential approach to address the aforesaid complications. The current study aims at the production of recombinant endoglucanase (CelA) originated from the cellulosome of Clostridium thermocellum in Escherichia coli (E. coli), followed by immobilization using modified regenerated cellulose (RC) membranes. The surface modification of RC membranes was performed in two different ways: one to generate the immobilized metal ion affinity membranes RC-EPI-IDA-Co2+ (IMAMs) for coordination coupling and another to develop aldehyde functional group membranes RC-EPI-DA-GA (AMs) for covalent bonding. For the preparation of IMAMs, cobalt ions expressed the highest affinity effect compared to other metal ions. Both enzyme-immobilized membranes exhibited better thermal stability and maintained an improved relative activity at higher temperatures (50–90 °C). In the storage analysis, 80% relative activity was retained after 15 days at 4 °C. Furthermore, the IMAM- and AM-immobilized CelA retained 63% and 53% relative activity, respectively, after being reused five times. As to the purification effect during immobilization, IMAMs showed a better purification fold of 3.19 than AMs. The IMAMs also displayed better kinetic parameters, with a higher Vmax of 15.57 U mg−1 and a lower Km of 36.14 mg mL−1, than those of AMs. The IMAMs were regenerated via treatment with stripping buffer and reloaded with enzymes and displayed almost 100% activity, the same as free enzymes, up to 5 cycles of regeneration.

Novel bi-functional thermostable chimeric enzyme for feasible xylo-oligosaccharides production from agro-industrial wastes

Arabinoxylan is a sustainable source for xylo-oligosaccharide production, as agro-industrial wastes are an abundant and the industrial interest for these prebiotics is raising due to their bioactivities for food and cosmetics. However, enzyme production costs and thermostability still represent a drawback. The main enzymes required for hydrolysis are endoxylanase and arabinofuranosidase, where GH 10 endoxylanase presents great thermostability and activity, and GH 62 enzymes are the arabinofuranosidases with the highest specificity but poor thermoresistance. In this sense, the fusion of these enzymes can generate a more stable enzyme with multiple catalytic domains in a single batch. The present study designed a bifunctional enzyme combining a GH 10 endoxylanase and a GH 62 arabinofuranosidase to enhance activity and thermostability, thus increasing the feasibility of xylo-oligosaccharides production from wastes. The chimera showed superior specificity for arabinoxylan and halotolerance than the parental enzymes and higher efficiency than commercial cocktail. Thermostability improved especially for the arabinofuranosidase module, which increased 10 °C, enabling a process at higher temperatures and lower enzyme loads, which increases efficiency and decreases contamination. Calcium and manganese enhanced thermostability and activity. Xylobiose and xylotriose yields improved by 25%, and up to 7 g/L XOS was achieved from corn hull arabinoxylan hydrolysis.

High cellulase‐free xylanases production by Moesziomyces aphidis using low‐cost carbon and nitrogen sources

AbstractBackgroundEnzymes involved in xylan hydrolysis have several industrial applications. Selection of efficient microbial hosts and scalable bioreaction operations can lower enzyme production costs and contribute to their commercial deployment. This work aims at investigating the Moesziomyces aphidis yeast cultivation conditions that deliver maximal xylanase titres, yields and productivities using low‐cost nitrogen (N) and carbon (C) sources.ResultsNaNO3 and KNO3 supplementation improved xylanase production 2.9‐ and 2.7‐fold (against 67.2 U mL−1), respectively, using xylan as C source. Interestingly, the use of KNO3, instead of NaNO3, results in 2‐ to 3‐fold higher specific activity, highlighting the potassium ion role. In addition, this study investigates synergetic effects on using ionic and organic N sources. A 4.9‐fold increase in xylanase production, with high specific activity, is attained combining KNO3 and corn steep liquor (CSL). Exploring the previous findings, this study reports one of the highest extracellular xylanase production titres (864.7 U mL−1) by yeasts, using a media formulation containing dilute‐acid pre‐treated brewery spent grains (BSG), as C source and inducer, supplemented with KNO3 and CSL. Replacement of dilute‐acid pre‐treatmed BSG by untreated BSG had low impact on xylanase production, of only 6%.ConclusionEfficient production of M. aphidis xylanolytic enzymes, using low‐cost N and C sources, is attractive for deployment of on‐site enzyme production targeting different biotechnological applications under circular economy and biorefinery concepts. Potential xylanases end‐users include industries such as brewing (using BSG as substrate for enzyme production), pulp and paper (benefiting from the cellulase‐free xylanase activity) or lignocellulosic ethanol (for cellulase supplementation). © 2022 Society of Chemical Industry (SCI).

Enhanced Cellulase Production by Talaromyces amestolkiae CMIAT055 Using Banana Pseudostem

Cellulases are a complex of enzymes necessary for the complete solubilization of cellulose in sugars, thus playing a key role in the natural carbon cycle through the hydrolysis of lignocellulosic structures. The aim of this study was to evaluate the increase in the capacity of Talaromyces amestolkiae CMIAT 055 to produce cellulases by optimizing the components of the culture medium containing banana pseudostem as an inducer, as well as in different agitation configurations in a bioreactor. Optimization was performed through statistical experimental design (Plackett–Burman and DCCR), a study of pH control in bioreactors, and a study of the agitation system by comparing impellers with different flow profiles in the liquid medium. For this purpose, a wild strain of Talaromyces amestolkiae CMIAT 055 was used. In the Plackett–Burman and DCCR statistical design, four components of the culture medium were significant and optimized for greater synthesis of FPase: banana pseudostem, CaCl2, KH2PO4, and urea. In bioreactors tests, these parameters were beneficial for greater enzyme activities: maintenance of pH at 5.0, use of Pitched blade impeller, and rotation speed at 300 rpm. Comparing the first test using banana pseudostem in an Erlenmeyer flask to the last fermentation process in bioreactors, it was observed that the total cellulase activity increased from 424.7 to 2172.8 FPU/L. This fact showed that the strategies adopted in this study are a pertinent way to reduce the cost of enzyme production through the use of lignocellulosic materials.Graphical Abstract

Underutilized Lignocellulosic Waste as Sources of Feedstock for Biofuel Production in Developing Countries

The need for a reliable and sustainable energy source, stability in energy price and solution to environmental challenges of fossil fuel has led to searching for an alternative energy source to fossil fuel. Several alternative sources have been developed over time, but they are limited in one form or another. However, biofuel such as bioethanol has been identified as a superb alternative with superior properties to fossil fuel. One major challenge with biofuel is the high production cost resulting from feedstock, which may also serve as a food source. In order to address this challenge, research is focused on searching for cheap and sustainable feedstock for biofuel production. Currently, attention is on lignocellulosic waste as feedstock with a keen interest in developing the most appropriate technique for processing it to bioethanol, especially in developing countries, which is the focus of this review. This review involves converting lignocellulosic waste to bioethanol and the pretreatment steps involved as well as its challenges, prospect and economic aspect. Among the pretreatment steps reported, biological treatment remains outstanding but with a few challenges which can be managed. Biofuel has come to stay in developing countries with lots of opportunities that favours its production cost. Although the high cost of enzyme production has been identified as a challenge to the economic viability of lignocellulosic bioethanol, there is hope that developing an efficient bio-system for simultaneous saccharification and fermentation (SSF) and consolidated biomass processing may help circumvent the challenge. In conclusion, the effective utilization of lignocellulosic waste in an efficient biocatalyst system can serve as an economically viable means to overcome the challenge posed by fossil fuel.

Lipases: Sources of Acquisition, Ways of Production, and Recent Applications

Enzymes are extensively used in biotechnological processes in several areas of industry. They are sustainable and safe, and their specificity is another characteristic that improves the performance in the process. Among enzymes, lipase is relevant due to the ability to play different roles in the industry and the possibility of collecting them from microbial sources that are found in industrial residues. This can reduce the costs of enzyme production. In relation to that, lipase immobilization is an interesting process that allows the enzymes to be reused and improves enzyme robustness. Among them, the cross-linked enzyme aggregates (CLEAs) methodology is attractive due to its simplicity, low cost (given the absence of support), and greater interaction with the substrate. Thus, in this review, we discussed the potential of lipase. We reviewed the traditional and new sources of obtaining lipases, along with the ways of improving production, activity, and application in the 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.

Production of poly (l-lactide)-degrading enzyme by Actinomadura keratinilytica strain T16-1 under solid state fermentation using agricultural wastes as substrate.

Poly (l-lactide) (PLLA) is an aliphatic polyester that can be obtained from renewable resources and degraded by various microorganisms. In previous reports, Actinomadura keratinilytica strain T16-1 demonstrated high ability to degrade PLLA under various conditions. PLLA-degrading enzyme production under solid state fermentation has been sparsely studied. PLLA-degrading enzyme production by A. keratinilytica strain T16-1 was investigated using agricultural wastes as substrate under solid state fermentation (SSF). Three agricultural wastes as soybean meal, cassava chips and duckweed were tested as substrates for PLLA-degrading enzyme production by statistical methods using mixture design. Results revealed that using duckweed as the substrate gave the highest enzyme production (138.66 ± 13.57 U/g dry substrate). Maximum enzyme activity of 391.24 ± 15.57 U/g dry substrate was obtained under 10g duckweed, 10% inoculum size, 7days of cultivation time, pH 7.0, 2.8% PLLA powder, and 60% moisture content at 45°C. It can be concluded that duckweed is an inexpensive substrate, which reduces the costs of PLLA-degrading enzyme production, as an alternative to effective water weed management.

Bacillus amyloliquefaciens 35 M can exclusively produce and secrete proteases when cultured in soybean-meal-based medium

Some microbial strains are ideal producers of extracellular enzymes that can be used in various industries. However, in many fields, especially in the pharmaceutical field, these enzymes need to be recovered and purified through multistep processes and tedious procedures before they can be used. The recovery process is difficult and increases the cost of enzyme production. Therefore, reducing purification steps will greatly benefit the utilization of microbial enzymes. The 35 M strain of Bacillus amyloliquefaciens, which has high extracellular protease production, was isolated from a phosphate mine. When cultured in a medium with soybean meal as the main component, the maximum activity of extracellular protease reached 16,992 U/mL. SDS-PAGE showed that there were two main proteins in the fermentation supernatant, with a paucity of other defined protein bands. Mass spectrometry and zymogram analysis showed that the two main bands were two proteases, corresponding to alkaline protease (AprM) and neutral protease (NprM), respectively. Gene cloning, sequencing, and further comparisons were used to confirm AprM and NprM correspond to these proteases from B. amyloliquefaciens. Notably, SDS-PAGE and zymogram analysis showed that NprM had obviously higher catalytic efficiency toward casein than did AprM. Strain 35 M is a promising protease producer with great potential for applications in industrial protease production. Additionally, this study demonstrates strain 35 M may be particularly well suited to use in degrading anti-nutritional factors in soybean meal, so as to improve the nutritional value of soybean meal.

Production of Thermophilic Chitinase by Paenibacillus sp. TKU052 by Bioprocessing of Chitinous Fishery Wastes and Its Application in N-acetyl-D-glucosamine Production.

The bioprocessing of chitinous fishery wastes (CFWs) to chitinases through fermentation approaches has gained importance owing to its great benefits in reducing the enzyme production cost, and utilizing chitin waste. In this work, our study of the chitinase production of Paenibacillus sp. TKU052 in the presence of different kinds of CFWs revealed a preference for demineralized crab shells powder (deCSP); furthermore, a 72 kDa chitinase was isolated from the 0.5% deCSP-containing medium. The Paenibacillus sp. TKU052 chitinase displayed maximum activity at 70 °C and pH 4–5, while Zn2+, Fe3+, Triton X-100, Tween 40, and SDS exerted a negative effect on its activity, whereas Mn2+ and 2-mercaptoethanol were found to potentially enhance the activity. Among various kinds of polysaccharide, Paenibacillus sp. TKU052 chitinase exhibited the best catalytic activity on colloidal chitin (CC) with Km = 9.75 mg/mL and Vmax = 2.43 μmol/min. The assessment of the hydrolysis of CC and N-acetyl chitooligosaccharides revealed that Paenibacillus sp. TKU052 chitinase possesses multiple catalytic functions, including exochitinase, endochitinase, and N-acetyl-β-D-glucosaminidase activities. Finally, the combination of Paenibacillus sp. TKU052 chitinase and Streptomyces speibonae TKU048 N-acetyl-β-D-glucosaminidase could efficiently convert CC to N-acetyl-D-glucosamine (GlcNAc) with a production yield of 94.35–98.60% in 12–24 h.