Machine learning prediction of novel pectinolytic enzymes in Aspergillus niger through integrating heterogeneous (post-) genomics data

Pectinolytic enzymes are a variety of enzymes involved in breaking down pectin, a complex and abundant plant cell-wall polysaccharide. In nature, pectinolytic enzymes play an essential role in allowing bacteria and fungi to depolymerize and utilize pectin. In addition, pectinases have been widely applied in various industries, such as the food, wine, textile, paper and pulp industries. Due to their important biological function and increasing industrial potential, discovery of novel pectinolytic enzymes has received global interest. However, traditional enzyme characterization relies heavily on biochemical experiments, which are time consuming, laborious and expensive. To accelerate identification of novel pectinolytic enzymes, an automatic approach is needed. We developed a machine learning (ML) approach for predicting pectinases in the industrial workhorse fungus, Aspergillus niger. The prediction integrated a diverse range of features, including evolutionary profile, gene expression, transcriptional regulation and biochemical characteristics. Results on both the training and the independent testing dataset showed that our method achieved over 90 % accuracy, and recalled over 60 % of pectinolytic genes. Application of the ML model on the A. niger genome led to the identification of 83 pectinases, covering both previously described pectinases and novel pectinases that do not belong to any known pectinolytic enzyme family. Our study demonstrated the tremendous potential of ML in discovery of new industrial enzymes through integrating heterogeneous (post-) genomimcs data.

Pulp Enhancement of Oil Palm Empty Fruit Bunches (OPEFBs) via Biobleaching by Using Xylano-Pectinolytic Enzymes of Bacillus amyloliquefaciens ADI2

The present work reports the biobleaching effect on OPEFB pulp upon utilisation of extracellular xylano-pectinolytic enzymes simultaneously yielded from Bacillus amyloliquefaciens ADI2. The impacts of different doses, retention times, pH, and temperatures required for the pulp biobleaching process were delineated accordingly. Here, the OPEFB pulp was subjected to pre-treatment with xylano-pectinolytic enzymes generated from the same alkalo-thermotolerant isolate that yielded those of higher quality. Remarkable enhanced outcomes were observed across varying pulp attributes: for example, enzyme-treated pulp treated to chemical bleaching sequence generated improved brightness of 11.25%. This resulted in 11.25% of less chlorine or chemical consumption required for obtaining pulp with optical attributes identical to those generated via typical chemical bleaching processes. Ultimately, the reduced consumption of chlorine would minimise the organochlorine compounds found in an effluent, resulting in a lowered environmental effect of paper-making processes overall as a consequence. This will undoubtedly facilitate such environmentally-friendly technology incorporation in the paper pulp industry of today.

The chimeric GaaR-XlnR transcription factor induces pectinolytic activities in the presence of D-xylose in Aspergillus niger

Aspergillus niger is a filamentous fungus well known for its ability to produce a wide variety of pectinolytic enzymes, which have many applications in the industry. The transcriptional activator GaaR is induced by 2-keto-3-deoxy-L-galactonate, a compound derived from D-galacturonic acid, and plays a major role in the regulation of pectinolytic genes. The requirement for inducer molecules can be a limiting factor for the production of enzymes. Therefore, the generation of chimeric transcription factors able to activate the expression of pectinolytic genes by using underutilized agricultural residues would be highly valuable for industrial applications. In this study, we used the CRISPR/Cas9 system to generate three chimeric GaaR-XlnR transcription factors expressed by the xlnR promoter by swapping the N-terminal region of the xylanolytic regulator XlnR to that of the GaaR in A. niger. As a test case, we constructed a PpgaX-hph reporter strain to evaluate the alteration of transcription factor specificity in the chimeric mutants. Our results showed that the chimeric GaaR-XlnR transcription factor was induced in the presence of D-xylose. Additionally, we generated a constitutively active GaaR-XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Proteomics analysis confirmed the production of several pectinolytic enzymes by ΔgaaR mutants carrying the chimeric transcription factor. This correlates with the improved release of D-galacturonic acid from pectin by the GaaR-XlnR V756F mutant, as well as by the increased L-arabinose release from the pectin side chains by both chimeric mutants under inducing condition, which is required for efficient degradation of pectin. Key points • Chimeric transcription factors were generated by on-site mutations using CRISPR/Cas9. • PpgaX-hph reporter strain allowed for the screening of functional GaaR-XlnR mutants. • Chimeric GaaR-XlnR induced pectinolytic activities in the presence of D-xylose.

Combined cellulolytic and pectinolytic enzymes to increase the polyphenol extractability of coffee husks

Coffee husks (CHs) is a high biological activity by-product of the coffee bean industry, but currently, in Vietnam, it has not been used properly in the food industry. This study was conducted to investigate the combined cellulolytic and pectinolytic enzymes to increase the polyphenol extractability of coffee husks. The red ripe Robusta coffee cherries from Dăklak province, Vietnam were blanched, peeled and the coffee husks was then extracted. The effects of pectinase content (from 0 to 800 U/gCHs), cellulase content (from 0 to 800 U/gCHs), pH (from 3.0 to 5.0), temperature (from 40◦C to 60◦C), CHs: solvent ratio (from 1: 6 to 1:12) and extracted time (from 60 minutes to 120 minutes) on soluble solid content (SSC), total polyphenol content (TPC) and antioxidant activity were investigated. The appropriate conditions for the treatment were pectinase dosage of 600 U/gCHs, cellulase dosage of 400 U/gCHs, pH 4.0, temperature 50◦C, CHs:soluble ratio = 1:10 and incubation time of 60 min under which the SSC, TPC and antioxidant activity of extract were highest by 60.60.4 g/100g dry weight of CHs, 200.5 gGAE /100g dry weight of CHs, 1.5 +/- 0.1 mMTE/g dry weight of extract (according to DPPH assay) and 1.5 +/- 0.1 mMTE/g dry weight of extract (according to DPPH assay). From this result, it can be seen that the extracted solution from coffee husk is a potential source for the production of soft drinks with high antioxidant activity.

Immobilization of Pectinolytic Enzymes on Nylon 6/6 Carriers

Pectinolytic enzymes are an important tool for sustainable food production, with a wide range of applications in food processing technologies as well as the extraction of bioactive compounds from pectin-rich raw materials. In the present study, we immobilized commercial pectinase preparation onto pellet and thread shaped nylon 6/6 carriers and assessed its stability and reusability. Five commercial pectinase preparations were tested for different pectin de-polymerizing activities (pectinase, polygalacturonase, and pectin lyase activities). Thereafter, Pectinex® Ultra Tropical preparation, exhibiting the highest catalytic activities among the studied preparations (p < 0.0001), was immobilized on nylon 6/6 using dimethyl sulfate and glutaraldehyde. The immobilization yield was in accordance with the carrier surface area available for enzyme attachment, and it was 1.25 ± 0.10 U/g on threads, which was over 40 times higher than that on pellets. However, the inactivation of immobilized enzymes was not dependent on the shape of the carrier, indicating that the attachment of the enzymes on the surface of nylon 6/6 carriers was similar. The half-life of enzyme inactivation fast phase at 4 °C was 12.8 days. After 5 weeks, the unused threads retained 63% of their initial activity. Reusability study showed that after 20 successive cycles the remaining activity of the immobilized pectinase was 22%, indicating the good prospects of reusability of the immobilized enzyme preparations for industrial application.

Effect of Pectinolytic Enzyme Pretreatment on the Clarification of Cranberry Juice by Ultrafiltration

Cranberries, mainly processed as juice, have garnered interest over the past decade due to their high content of phytochemical compounds related to promising health benefits. To meet consumer expectations, a juice clarification step is usually incorporated to remove suspended solids. The use of pectinolytic enzyme and membrane processes are commonly applied to the production of clarified juices, but no studies have been done on cranberry juice. In this study, the effects of 60 (D60) and 120 min (D120) of depectinization by pectinolytic enzymes coupled to clarification by ultrafiltration (UF) (membrane molecular weight cut-off (MWCO) of 50, 100 and 500 kDa) was evaluated on the filtration performance, membrane fouling and cranberry juice composition. Compared to fresh juice, depectinization for 60 and 120 min reduced the UF duration by 16.7 and 20 min, respectively. The best filtration performance, in terms of permeate fluxes, was obtained with the 500 kDa MWCO UF membrane despite the highest total flux decline (41.5 to 57.6%). The fouling layer at the membrane surface was composed of polyphenols and anthocyanins. Compared to fresh juice, anthocyanin decreased (44% and 58% for D60 and D120, respectively) in depectinized juices whereas proanthocyanidin (PAC) content increased by 16%. In view of the industrial application, a 60 min depectinization coupled to clarification by a 500 kDa UF membrane could be viewed as a good compromise between the enhancement of filtration performance and the loss of polyphenols and their fouling at the membrane surface.