Combinatorial Assembly and Optimisation of Designer Cellulosomes - A Galactomannan Case Study

Background: Designer cellulosomes are self-assembled chimeric enzyme complexes that can be used to improve lignocellulosic biomass degradation. They are composed of a synthetic multimodular backbone protein, termed the scaffoldin, and a range of different chimeric docking enzymes that degrade polysaccharides. Over the years, several functional designer cellulosomes have been constructed. Since many parameters influence the efficiency of these multi-enzyme complexes, there is a need to optimise designer cellulosome architecture by testing combinatorial arrangements of docking enzyme and scaffoldin variants. However, the modular cloning procedures are tedious and cumbersome. Results: VersaTile is a combinatorial DNA assembly method, allowing the rapid construction and thus comparison of a range of modular proteins. Here, we present the extension of the VersaTile platform to facilitate the construction of designer cellulosomes. We have constructed a tile repository, composed of dockerins, cohesins, linkers, tags and enzymatically active modules. The developed toolbox allows us to efficiently create and optimise designer cellulosomes at an unprecedented speed. As a proof of concept, a trivalent designer cellulosome able to degrade the specific hemicellulose substrate, galactomannan, was constructed and optimised. The main factors influencing cellulosome efficiency were found to be the selected dockerins and linkers and the docking enzyme ratio on the scaffoldin. The optimised designer cellulosome was able to hydrolyse the galactomannan polysaccharide and release mannose and galactose monomers. Conclusion: We have eliminated one of the main technical hurdles in the designer cellulosome field and anticipate the VersaTile platform to be a starting point in the development of more elaborate multi-enzyme complexes.

Long-term warming and nitrogen fertilization affect C-, N- and P-acquiring hydrolase and oxidase activities in winter wheat monocropping soil

The enzymatic activities and ratios are critical indicators for organic matter decomposition and provide potentially positive feedback to carbon (C) loss under global warming. For agricultural soils under climate change, the effect of long-term warming on the activities of oxidases and hydrolases targeting C, nitrogen (N) and phosphorus (P) and their ratios is unclear, as well as whether and to what extend the response is modulated by long-term fertilization. A 9-year field experiment in the North China Plain, including an untreated control, warming, N fertilization, and combined (WN) treatment plots, compared the factorial effect of warming and fertilization. Long-term warming interacted with fertilization to stimulate the highest activities of C, N, and P hydrolases. Activities of C and P hydrolase increased from 8 to 69% by N fertilization, 9 to 53% by warming, and 28 to 130% by WN treatment compared to control, whereas the activities of oxidase increased from 4 to 16% in the WN soils. Both the warming and the WN treatments significantly increased the enzymatic C:N ratio from 0.06 to 0.16 and the vector length from 0.04 to 0.12 compared to the control soil, indicating higher energy and resource limitation for the soil microorganisms. Compared to WN, the warming induced similar ratio of oxidase to C hydrolase, showing a comparable ability of different microbial communities to utilize lignin substrates. The relationship analyses showed mineralization of organic N to mediate the decomposition of lignin and enzyme ratio in the long-term warming soil, while N and P hydrolases cooperatively benefited to induce more oxidase productions in the soil subject to both warming and N fertilization. We conclude that coupled resource limitations induced microbial acclimation to long-term warming in the agricultural soils experiencing high N fertilizer inputs.

Hydrolysate from Mussel Mytilus galloprovincialis Meat: Enzymatic Hydrolysis, Optimization and Bioactive Properties

Mussel production generates losses and waste since their commercialisation must be aligned with target market criteria. Since mussels are rich in proteins, their meat can be explored as a source of bioactive hydrolysates. Thus, the main objective of this study was to establish the optimal production conditions through two Box–Behnken designs to produce, by enzymatic hydrolysis (using subtilisin and corolase), hydrolysates rich in proteins and with bioactive properties. The factorial design allowed for the evaluation of the effects of three factors (hydrolysis temperature, enzyme ratio, and hydrolysis time) on protein/peptides release as well as antioxidant and anti-hypertensive properties of the hydrolysates. The hydrolysates produced using the optimised conditions using the subtilisin protease showed 45.0 ± 0.38% of protein, antioxidant activity via ORAC method of 485.63 ± 60.65 µmol TE/g of hydrolysate, and an IC50 for the inhibition of ACE of 1.0 ± 0.56 mg of protein/mL. The hydrolysates produced using corolase showed 46.35 ± 1.12% of protein, antioxidant activity of 389.48 ± 0.21 µmol TE/g of hydrolysate, and an IC50 for the inhibition of ACE of 3.7 ± 0.33 mg of protein/mL. Mussel meat losses and waste can be used as a source of hydrolysates rich in peptides with relevant bioactive properties, and showing potential for use as ingredients in different industries, such as food and cosmetics, contributing to a circular economy and reducing world waste.

Design of Stirred Tank Reactor for Cellulase Enzyme Rumen Liquid Based Bioethanol Production from Banana Rod

Due to pandemic, supply of alcohol as desinfectant really needed for nowadays. This research aims to determine the levels of bioethanol produced from banana rods by the hydrolysis process. The target to be achieved with the reactor design is to achieve high ethanol purity using a first order reaction kinetics approach. The study was carried out on various variables including fermentation time (6 - 30 hours), hydrolysis temperature (80 - 90 oC), and substrate enzyme ratio (1: 1 - 1: 3). While the parameters to be tested are glucose levels and bioethanol levels. The optimal effect of the variable on alcohol content is obtained when the alcohol content value is more than 17.2% with an enzyme ratio of 3 - 3.5 at a temperature of 94 - 96 oC for 35 - 40 hours. From the research, it was found that it is possible to enlarge the reactor up to 200 times by using a ratio to obtain the ideal stirred tank reactor dimensions with a conversion of 97.15%.

Biochemical Mechanism of Insecticide Resistance in Malaria Vector, Anopheles gambiae s.l in Nigeria

Background: Malaria is a parasitic vector-borne disease endemic in the tropical and subtropical countries of the world. The aim of this study was to investigate the current activities of the detoxification enzymes in resistant and susceptible Anopheles gambiae s.l. in northern Nigeria. Methods: Anopheles larvae were collected from northeast and northwestern Nigeria between Aug and Nov 2018. Biochemical analyses was carried out on the mosquitoes exposed to various insecticides (deltamethrin, DDT, bendiocarb, malathion) to measure and compare the enzymatic activities of the major detoxification enzymes (P450, GSTs, Esterase). Results: High levels of resistance was observed; DDT 37%-53% (95%, CI: 29-61), bendiocarb 44%-55% (CI: 39- 60) and deltamethrin 74%-82% (CI: 70-86). However, these mosquitoes were found to be susceptible to malathion 99%-100% (CI: 98-100). The P450 and GSTs enzymes were found to be elevated in the resistant mosquitoes exposed to deltamethrin (1.0240±0.1902); (1.3088±1.2478), DDT (1.7703±1.4528); (1.7462±0.9418) and bendiocarb (1.1814±0.0918); (1.4479±1.0083) compared to the Kisumu strain (0.764±0.4226); (0.6508±0.6542), (0.3875±0.3482); (0.4072±0.4916) and (0.6672±0.3949); (0.7126±0.7259) at P<0.05. Similarly, the resistant mosquitoes expressed increased activity to esterase (0.7606±1.1477), (0.3269±1.1957) and (2.8203±0.6488) compared to their susceptible counterpart (0.6841±0.7597), (0.7032±0.5380) and (0.6398±0.4159) at P<0.05. The enzyme ratio was found to be: P450 (1.341, 4.568 and 1.77); GSTs (2.011, 4.288 and 2.031); Esterases (1.111, 0.469 and 4.408). One way Anova and single sample t-test were also conducted to determine the effect of the enzymes on the resistant and susceptible strains. Conclusion: High level of insecticide resistance was observed with significant elevation of detoxification enzymes activities in the resistant mosquitoes.

A DNA nanopillar as a scaffold to regulate the ratio and distance of mimic enzymes for an efficient cascade catalytic platform

A rigid 3D DNA nanopillar was used to investigate the influence of spatial organization on the cascade activity in multienzyme systems, realizing controllable regulation of the mimic enzyme ratio and spacing for efficient cascade catalytic platform.

Response Surface Optimization and Impact of Immobilized Enzymes Naringianse and Tannase on the Quality Parameters of Citrus maxima Juice

Pomelo has been reported as a rich source of flavanone glycoside with antioxidants and exhibits favorable health effects such as antimicrobial, anti-inflammatory, antiatherogenic, antitumor, and anti-clotting activity. Despite all the beneficial health impacts of Citrus maxima, it still has lower commercial value because of its juice's bitterness due to the presence of naringin and tannic acid. Therefore, an attempt has been made for the cost-effective and economic debittering process using naringinase and tannase enzymes. The 17 experiments were planned according to RSM, BBD to analyze the effect of independent variables with three levels of each, i.e., Enzyme ratio ((Naringinase: tannase) (100:0, 50:50, 0:100)), incubation temperature (30°C, 40°C, 50°C) and incubation time (2, 3, 4 hrs) on physicochemical quality of Citrus maxima Juice. The study's result indicated that independent variables affected the responses (pH, TSS, TA, Naringin content, Tannin content, TPC, and Vitamin C content). Optimization was done using Design Expert 10.0.1 software to debitter and clarify citrus maxima juice by immobilized enzymes. The optimum values were found to be 54.55, 50°C, and 4 hrs. The values for pH, TSS, TA, Naringin content, Tannin content, TPC and Vitamin C content were found to be 3.17, 6.256 °Brix, 0.885 %citric acid , 220.549 μg/ml ,0.311mg/ml, 1256.721 mg GAE/L,30.309 mg/100ml respectively. From the study, it could be concluded that the maximum debittering and clarification of citrus maxima juice could be done under processing conditions, i.e., enzyme ratio 50:50, incubation temperature 50°C and incubation time 4 hrs.

Design of Stirred Tank Reactor for Bioetanol Production From Banana Rod Using Cellulase Enzymes Cow's Liquid Rumen

Due to pandemic, supply of alcohol as desinfectant really needed for nowadays. This research aims to determine the levels of bioethanol produced from banana rods by the hydrolysis process. The target to be achieved with the reactor design is to achieve high ethanol purity using a first order reaction kinetics approach. The study was carried out on various variables including fermentation time (6 - 30 hours), hydrolysis temperature (80 - 90 oC), and substrate enzyme ratio (1: 1 - 1: 3). While the parameters to be tested are glucose levels and bioethanol levels. The optimal effect of the variable on alcohol content is obtained when the \alcohol content value is more than 17.2% with an enzyme ratio of 3 - 3.5 at a temperature of 94 - 96 oC for 35 - 40 hours. From the research, it was found that it is possible to enlarge the reactor up to 10000 times by using a ratio ratio to obtain the ideal stirred tank reactor dimensions with a conversion of 97.15%.

Screening of factors influencing the hydrolysis reaction of chicken leg cartilage with Protamex enzyme as a catalyst

The objective of this research was to use Protamex enzyme as a catalyst to hydrolyze chicken leg cartilage for production of amino acid hydrolysate. The proximate composition of chicken cartilage was analyzed. The results indicated that the major components in the dry matter of chicken leg cartilage were lipid (19.72 ± 0.05%) and protein (13.34 ± 0.08%). The main parameters affecting the hydrolysis reaction of chicken leg cartilage with Protamex enzyme were selected for investigation: reaction temperature (oC), pH, enzyme ratio (%, based on the weight of substrate), reaction time (min), and the ratio of reaction volume (v/v). The significant difference was analyzed by ANOVA One-Way to identify the optimal point of each parameter toward amino acid yield. The results revealed that the hydrolysis degree and amino acid yield reached the maximal values of 22.93 ± 4.01% and 30.25 ± 1.86%, respectively, when the reaction temperature, pH, enzyme ratio, reaction time, and ratio of reaction volume (v/v) were 40oC, 4, 0.52%, 40 min, and 1/18, respectively.

Kinetics of Enzymatic Hydrolysis of Passion Fruit Peel using Cellulase in Bio-ethanol Production

This research aims to study the hydrolysis of passion fruit peel using cellulase and its evaluation for ethanol production. Passion fruit peel is a fruit processing waste that has not been utilized properly. Passion fruit peel contains holo-cellulose (64% w/w), which can be converted into ethanol through hydrolysis followed by fermentation. Hydrolysis using cellulase is more efficient and its fermentation using yeast to produce ethanol is common. The hydrolysis is carried out at various enzyme ratios (3, 5, 7, and 9% v/v) and at temperature 30 oC, material concentration 5 g/100 mL, pH 4-5, and shaking speed 160 rpm. The kinetics chosen were heterogeneous models; they were the fractal model by Valjamae and Kopelman. Before being hydrolyzed, the essential oil and pectin in passion fruit peel were extracted, because the compositions were quite high; the results were around 16.23 and 11.36% w/w, respectively. The effect of the enzyme ratio to the sugar concentration by hydrolysis is very significant. At 9 h, the glucose concentration reached 45.38, 51.86, 60.50, 66.00 g/L at various enzyme ratios of 3, 5, 7, 9% v/v. During the hydrolysis, the glucose concentration continues to increase and starts to decrease after 9 h. Hydrolyzate solution fermentation obtained from hydrolysis in various enzyme ratios showed consistent results; the higher the enzyme ratio and glucose, and the higher the ethanol will be (5.6, 6.8, 7.6, and 8.9% v/v). The kinetics model by Valjamae is more appropriate to describe the enzymatic hydrolysis mechanism of passion fruit peel than Kopelman. The fractal exponent values obtained from Valjamae and Kopelman models were 0.28 and 0.27. In Valjamae model, the enzyme ratio rises, from 3 to 9% v/v, the rate constant rises from 0.22 to 0.53 1/h. In Kopelman model, the rate constant rises too, from 0.21 to 0.51 1/h.Keywords: bio-ethanol; cellulase; enzymatic hydrolysis; fractal kinetic; passion fruit peel