RESUMEN

Laccases are multi-copper oxidases that are usually composed of three Cu-oxidase domains. Domains one and three house the copper binding sites, and the second domain is involved in forming a substrate-binding cleft. However, Streptomyces species are found to have small laccases (SLAC) that lack one of the three Cu-oxidase domains. This type of SLAC with interesting lignocellulose bioconversion activities has not been reported in Aspergillus niger. In our research, we explored the expression and engineering of the SLAC from Streptomyces leeuwenhoekii C34 in A. niger. Genes encoding two versions of the SLAC were expressed. One encoding the SLAC in its native form and a second encoding the SLAC fused to two N-terminal CBM1 domains. The latter is a configuration also known for specific yeast laccases. Both SLAC variants were functionally expressed in A. niger as shown by in vitro activity assays and proteome analysis. Laccase activity was also analyzed toward bioconversion of lignocellulosic rice straw. From this analysis it was clear that the SLAC activity improved the efficiency of saccharification of lignocellulosic biomass by cellulase enzyme cocktails.

RESUMEN

The glycoside hydrolase family 3 (GH3) ß-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 ß-glucosidases. Two distinct configurations were identified in fungal GH3 ß-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in ß-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger ß-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric ß-glucosidases enzymes for industrial application.

Asunto(s)

Ingeniería de Proteínas , Streptomyces , beta-Glucosidasa , Streptomyces/enzimología , Streptomyces/genética , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismo , beta-Glucosidasa/química , Ingeniería de Proteínas/métodos , Biotecnología/métodos , Aspergillus niger/enzimología , Aspergillus niger/genética , Dominios Proteicos , Aspergillus/enzimología , Aspergillus/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Dominio Catalítico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química

RESUMEN

In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes.

Asunto(s)

Aspergillus niger , alfa-Amilasas , alfa-Amilasas/metabolismo , Aspergillus niger/metabolismo , Almidón/química , Hidrólisis , Metabolismo de los Hidratos de Carbono

RESUMEN

Enzymatic degradation of abundant renewable polysaccharides such as cellulose and starch is a field that has the attention of both the industrial and scientific community. Most of the polysaccharide degrading enzymes are classified into several glycoside hydrolase families. They are often organized in a modular manner which includes a catalytic domain connected to one or more carbohydrate-binding modules. The carbohydrate-binding modules (CBM) have been shown to increase the proximity of the enzyme to its substrate, especially for insoluble substrates. Therefore, these modules are considered to enhance enzymatic hydrolysis. These properties have played an important role in many biotechnological applications with the aim to improve the efficiency of polysaccharide degradation. The domain organization of glycoside hydrolases (GHs) equipped with one or more CBM does vary within organisms. This review comprehensively highlights the presence of CBM as ancillary modules and explores the diversity of GHs carrying one or more of these modules that actively act either on cellulose or starch. Special emphasis is given to the cellulase and amylase distribution within the filamentous microorganisms from the genera of Streptomyces and Aspergillus that are well known to have a great capacity for secreting a wide range of these polysaccharide degrading enzyme. The potential of the CBM and other ancillary domains for the design of improved polysaccharide decomposing enzymes is discussed.

RESUMEN

Aims@#To identify mold contaminant on salted fish, from two different market locations (Kenjeran market, Surabaya and Beringharjo market, Yogyakarta). Furthermore, levels of AFB1 (aflatoxin B1) in salted fish samples were assayed. @*Methodology and results@#The samples were cultivated on DRBC (Dichloran Rose Bengal Chloramphenicol Agar) and DG-18 (Dichloran (18%) Glycerol Agar) medium for enumeration, then transferred on MEA (Malt Extract Agar) medium for isolation and identification, followed by ELISA test to measure the AFB1 level. Meanwhile aflatoxin biosynthesis correlated genes (i.e. aflR, nor-1 and omtB genes) were identified using Polymerase Chain Reaction (PCR) method. The results showed that Aspergillus tamarii and A. flavus being contaminant on salted fish along with A. sydowii, A. niger, A. versicolor, Penicillium citrinum, and P. chrysogenum. Rhizopus sp. contamination was also found. AFB 1 was positively detected in all of samples with the highest concentration measured was 75.81 μg/kg which belong to Lidah salted fish and the lowest concentration measured was 4.33 μg/kg which belong to Rese salted fish. The suspected A. flavus and A. tamarii isolated from salted fish was positively detected in the presence of aflR, nor-1 and omtB genes. @*Conclusion, significance and impact of study@#Mold contamination was detected in salted fish from two different markets and all of those samples were contaminated by AFB1. These can be important information related to food safety aspect for salted fish.